Regulated apoptosis

ABSTRACT

We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins and disclose methods and materials for using that procedure to regulatably initiate cell-specific apoptosis (programmed cell death) in genetically engineered cells

TECHNICAL FIELD

[0001] This invention concerns materials, methods and applicationsrelating to the oligomerizing of chimeric proteins with a dimeric ormultimeric, preferably non-peptidic, organic molecule. Aspects of theinvention are exemplified by recombinant modifications of host cells andtheir use in gene therapy or other applications of inducible geneexpression.

INTRODUCTION

[0002] Biological specificity usually results from highly specificinteractions among proteins. This principle is exemplified by signaltransduction, the process by which extracellular molecules influenceintracellular events. Many pathways originate with the binding ofextracellular ligands to cell surface receptors. In many cases receptordimerization leads to transphosphorylation and the recruitment ofproteins that continue the signaling cascade. The realization thatmembrane receptors could be activated by homodimerization resulted fromthe observation that receptors could be activated by antibodies thatcross linked two receptors. Subsequently, many receptors were found toshare those properties. The extracellular and transmembrane regions ofmany receptors are believed to function by bringing the cytoplasmicdomains of the receptors in dose proximity by a ligand-dependentdimerization or oligomerization, while the cytoplasmic domains of thereceptor convey specific signals to internal compartments of the cell.

[0003] Others have investigated ligand-receptor interactions indifferent systems. For example, Clark, et al., Science (1992) 258, 123describe cytoplasmic effectors of the B-cell antigen receptor complexDurand, et al., Mol. Cell. Biol. (1988) 8, 1715, Verweij, et al., J.Biol. Chem. (1990) 265, 15788 and Shaw, et al., Science (1988) 241, 202report that the NF-AT-directed transcription is rigorously under thecontrol of the antigen receptor. Inhibition of NF-AT-directedtranscription by cyclosporin A and FK506 is reported by Emmel, et al.,Science (1989) 246, 1617 and Flanagan, et al., Nature (1991) 352, 803.Durand, et al., Mol. Cell. Biol. (1988) 8, 1715 and Mattila, et al.,EMBO J. (1990) 9, 4425 describe the NF-AT binding sites. Referencesdescribing the ζ chain include Orloff, et al., Nature (1990) 347,189-191; Kinet, et al., Cell (1989) 57, 351-354; Weissman, et al., Proc.Natl. Acad. Sci. USA (1988) 85, 9709-9713 and Lanier, Nature (1989) 342,803-805. A CD4 immunoadhesin is described by Byrn, et al. Nature (1990)344, 667-670. A CD8-ζ-fused protein is described by Irving, et al., Cell(1992) 64, 891. See also, Letourner and Klausner, Science (1992) 255,79.

[0004] Illustrative articles describing transcriptional factorassociation with promoter regions and the separate activation and DNAbinding of transcription factors include: Keegan et al., Nature (1986)231, 699; Fields and Song, ibid (1989) 340, 245; Jones, Cell (1990) 61,9; Lewin, Cell (1990) 61, 1161; Ptashne and Gann, Nature (1990) 346,329; Adams and Workman, Cell (1993) 72, 306.

[0005] Illustrative articles describing vesicle targeting and fusioninclude: Sollner et al. (1993) Nature 362, 318-324; and Bennett andScheller (1993) Proc. Natl. Acad. Sci. USA 90, 2559-2563.

[0006] Illustrative articles describing regulated protein degradationinclude: Hochstrasser et al (1990) Cell 61, 697; Scheffner, M. et al(1993) Cell 75, 495; Rogers et al (1986) Science 234, 361-368.

[0007] Illustrative publications providing additional informationconcerning synthetic techniques and modifications relevant to FK506 andrelated compounds include: GB 2 244 991 A; EP 0 455 427 A1; WO 91/17754;EP 0 465 426 A1, U.S. Pat. No. 5,023,263 and WO 92/00278.

[0008] Illustrative publications concerning the Fas antigen, p55 TNFreceptor (hereinafter “TNF receptor”) and/or apoptosis include: Itoh, etal. (1991) Cell 66, 233-243; Nagata, et al., European Patent ApplicationPublication No. 510 691 (1992); Suda et al, Cell (1993), 75(6), 1169-78;Oehm, et al., J Biological Chem. (1992) 267(15), 10709-10715; and Wongand Goeddel, J Immunol (1994),152(4), 1751-5.

[0009] Illustrative discussion of methods and materials for gene therapyis found in Chapter 28 of Watson, Gilman, Witkowski and Zoller,RECOMBINANT DNA, 2d edition (WH Freeman & Co, 1992) and in referencescited in the bibliography therein especially on pp 564-565.

[0010] However, as will be clear from this disclosure, none of theforegoing authors describe or suggest the present invention. Ourinvention, which is disclosed in detail hereinafter, involves agenerally applicable method and materials for utilizing proteinhomodimerization, heterodimerization and oligomerization in livingcells. (As used herein, the terms oligomer, oligomerize andoligomerization encompass dimers, trimers and higher order oligomers andtheir formation.) Chimeric responder proteins are intracellularlyexpressed as fusion proteins with a specific receptor domain. Treatmentof the cells with a cell permeable multivalent ligand reagent whichbinds to the receptor domain leads to dimerization or oligomerization ofthe chimera. In analogy to other chimeric receptors (see e.g. Weiss,Cell (1993) 73, 209), the chimeric proteins are designed such thatoligomerization triggers cell death, and in certain embodiments,optional other subsequent events, e.g. the propagation of anintracellular signal via subsequent protein-protein interactions andthereby the activation of a specific subset of transcription factors.The initiation of transcription can be detected using a reporter geneassay. Intracellular crosslinking of chimeric proteins by syntheticligands has potential in basic investigation of a variety of cellularprocesses, in regulatably initiating cell death in engineered cells andin regulating the synthesis of proteins of therapeutic or agriculturalimportance. Furthermore, ligand mediated oligomerization now permitsregulated gene therapy. In so doing, it provides a fresh approach toincreasing the safety, expression level and overall efficacy obtainedwith gene therapy.

SUMMARY OF THE INVENTION

[0011] This invention provides materials and methods for the geneticengineering of host cells to render the cells and their progenysusceptible, in a regulated fashion, to programmed cell death(apoptosis). This invention is useful as a means for eliminating apopulation of engineered cells, whether growing in culture or in vivo,and thus provides, inter alia, a fail-safe mechanism for geneticallyengineered cells used in gene therapy.

[0012] The invention involves novel chimeric (or “fused”) proteins, DNAconstructs encoding them, and ligand molecules capable of oligomerizingthe chimeric proteins. The chimeric proteins contain at least oneligand-binding (or “receptor”) domain fused to an action domain capableof initiating apoptosis within a cell, as described in detail below. Aswill also be described, the chimeric proteins may also containadditional domains. The chimeric proteins are recombinant in the sensethat the various domains are derived from different sources, and assuch, are not found together in nature (i.e., are heterologous);

[0013] This invention provides DNA molecules (“constructs”) which encodethe novel chimeric proteins and which may be used for the geneticengineering of host cells. These constructs are recombinant in the sensethat the component portions, e.g. encoding a particular domain orexpression control sequence, are not found directly linked to oneanother in nature. Also provided are methods and compositions forproducing and using the modified cells.

[0014] To produce the modified cells one introduces DNA encoding thedesired chimera(s) into selected host cells. This may be accomplishedusing conventional vectors (various examples of which are commerciallyavailable) and techniques. If desired, the modified cells may then beselected, separated from other cells and cultured, again by conventionalmethods.

[0015] The oligomerizing ligands useful in practicing this invention arecapable of binding to two (or more) of the receptor domains, i.e. to twoor more chimeric proteins containing such receptor domains. Theoligomerizing ligand may bind to the chimera in either order orsimultaneously, preferably with a Kd value below about 10⁻⁶, morepreferably below about 10⁻⁷, even more preferably below about 10⁻⁸, andin some embodiments below about 10⁻⁹ M. The ligand preferably is anon-protein and has a molecular weight of less than about 5 kDa. Thereceptor domains of the chimeric proteins so oligomerized may be thesame or different. The chimeric proteins are capable of initiatingapoptosis of their host cell upon exposure to the ligand, i.e.,following oligomerization of the chimera. Thus, apoptosis of geneticallyengineered cells of this invention occurs following exposure of thecells to a ligand capable of oligomerizing the chimera. Saiddifferently, genetically engineered cells of this invention containchimeric proteins as described above and are responsive to the presenceof a ligand which is capable of oligomerizing those chimera. Thatresponsiveness is manifested by the initiation of cell death.

[0016] The encoded chimeric protein may further comprise anintracellular targeting domain capable of directing the chimeric proteinto a desired cellular compartment. The targeting domain can be asecretory leader sequence, a membrane spanning domain, a membranebinding domain or a sequence directing the protein to associate withvesicles or with the nucleus, for instance.

[0017] The action domains of the chimeric proteins may be selected fromany of the proteins or protein domains (preferably of human origin orsequence) which trigger apoptosis upon crosslinking, including, forexample, the cytoplasmic domain of the Fas antigen.

[0018] As discussed in greater detail later, and by way of example, invarious embodiments of this invention the chimeric protein is capable ofbinding to an FK506-type ligand, a cyclosporin A-type ligand,tetracycline or a steroid ligand. Such binding leads to oligomerizationof the chimeric protein with other chimeric protein molecules which maybe the same or different.

[0019] In addition to the construct(s) encoding the chimera describedabove (the “primary” chimera), the cells may optionally further containadditional heterologous DNA constructs for the regulatable orconstitutive expression of one or more desired genes. For example, thecells may additionally contain one or more other constructs encodingoptional chimera, otherwise as described above but containing actiondomains which, upon ligand-induced oligomerization, trigger biologicalevents other than apoptosis. Such other action domains may be selectedfrom a broad variety of protein domains capable of effecting a desiredbiological result upon oligomerization of the chimeric protein(s). Forinstance, the action domain may comprise a protein domain such as a CD3zeta subunit capable, upon exposure to the ligand and subsequentoligomerization, of initiating a detectable intracellular signal; aDNA-binding protein such as GAL4; or a transcriptional activation domainsuch as VP16. Numerous other examples are provided herein. One exampleof a detectable intracellular signal is a signal activating thetranscription of a gene under the transcriptional control of atranscriptional control element (e.g. enhancer and/or promoter elementsand the like) which is responsive to the oligomerization. Preferably theligand(s) which oligomerize the primary chimera and lead to apoptosis donot cause oligomerization of the optional chimeric proteins. It isusually even more preferable that the ligand(s) which oligomerize theoptional chimera and effect the optional biological events, such asregulated gene transcription, do not lead to oligomerization of theprimary chimera or trigger apoptosis. The different sets of ligands arein that sense orthogonal.

[0020] As is discussed in greater detail later, in various embodimentsof is invention the chimeric proteins are capable of binding to anFK506-type ligand, a cyclosporin A-type ligand, tetracycline or asteroid ligand. Such binding leads to oligomerization of the chimericprotein molecules with other chimeric protein molecules which may be thesame or different.

[0021] Optionally the cells may contain still another recombinantconstruct or series of such construct(s), containing a target gene underthe transcriptional control of a transcriptional control element (e.g.promoter/enhancer) responsive to a signal triggered by ligand-mediatedoligomerization of optional chimeric proteins, i.e. to exposure of thecells to the relevant ligand. These constructs are recombinant in thesense that the target gene is not naturally under the transcriptionalcontrol of the responsive transcriptional control element.

[0022] Such an optional target gene construct may contain (a) atranscriptional control element responsive to the oligomerization of anoptional chimeric protein as described above, and (b) flanking DNAsequence from a target gene permitting the homologous recombination ofthe transcriptional control element into a host cell in association withthe target gene. In other embodiments the construct contains a desiredgene and flanking DNA sequence from a target locus permitting thehomologous recombination of the target gene into the desired locus. (Seee.g., Mansour et al, 1988, Nature 336, 348-352 and subsequent papers byM. Capecchi et al.) The construct may also contain the responsivetranscriptional control element, or the responsive element may beprovided by the locus. The target gene may encode, e.g., a surfacemembrane protein, a secreted protein, a cytoplasmic protein or aribozyme or an antisense sequence.

[0023] The constructs of this invention may also contain a selectablemarker permitting transfection of the constructs into host cells andselection of transfectants containing the construct. This inventionfurther encompasses DNA vectors containing the various constructsdescribed herein, whether for introduction of the constructs into hostcells in tissue culture or for administration to whole organisms forintroduction into cells in vivo. In either case the construct may beintroduced episomally or for chromosomal integration. The vector may bea viral vector, including for example an adeno-, adeno associated- orretroviral vector.

[0024] This invention further encompasses a chimeric protein encoded byany of our DNA constructs, as well as cells containing and/or expressingthem, including prokaryotic and eucaryotic cells and in particular,yeast, worm insect, mouse or other rodent, and other mammalian cells,including human cells, of various types and lineages, whether frozen orin active growth, whether in culture or in a whole organism containingthem.

[0025] To recap, this invention provides cells, preferably but notnecessarily mammalian, which contain a first DNA construct encoding aprimary chimeric protein comprising (i) at least one receptor domaincapable of binding to a selected oligomerizing ligand of this inventionand (ii) another protein domain, heterologous with respect to thereceptor domain but capable, upon oligomerization with one or more otherlike domains, of triggering apoptosis of the cells. Following exposureof the cells to the selected ligand, programmed cell death ensues.

[0026] In some embodiments, the cells, as just described, also containone or more optional DNA constructs encoding one or more chimericproteins comprising (i) at least one receptor domain capable of bindingto a selected oligomerizing ligand of this invention and (ii) anotherprotein domain, heterologous with respect to the receptor domain, butcapable, upon oligomerization of these optional chimera, of triggering(directly or indirectly) the activation of transcription of a targetgene under the transcriptional control of a transitional control elementresponsive to said oligomerization. The cells will usually also containa target gene under the expression control of a transitional controlelement responsive to said oligomerization ligand. Following exposure tothe selected ligand the target gene is expressed. Again, the ligandcapable of oligomerizing the primary chimera and the ligand(s) capableof oligomerizing the optional chimera should be orthogonal.

[0027] In other embodiments, the cells of this invention also contain aDNA construct encoding a first optional chimeric protein containing aDNA-binding domain and at least one receptor domain capable of bindingto a first selected ligand moiety. The cells further contain a secondoptional chimeric protein containing a transcriptional activating domainand at least one receptor domain capable of binding to a second selectedligand moiety (which may be the same or different from the firstselected ligand moiety). The cells additionally contain a DNA constructencoding a target gene under the transcriptional control of aheterologous transcriptional control sequence with a cognate bindingsite for the DNA-binding domain and which is responsive to thetranscriptional activating domain such that the cell expresses thetarget gene following exposure to an oligomerizing ligand containing theselected ligand moiety(ies).

[0028] DNA compositions useful for practicing aspects of the inventioninclude those which encode the optional chimera. Those compositionscomprise a first DNA construct encoding a chimeric protein comprising atleast one receptor domain, capable of binding to a selected ligand,fused to a heterologous additional protein domain capable of initiatinga biological process upon exposure to the oligomerizing ligand, i.e.upon oligomerization of the chimeric protein; and a second DNA constructencoding a target gene under the transcriptional control of atranscription control element responsive to the oligomerization ligand.

[0029] Another exemplary DNA composition useful in practicing aspects ofthis invention comprises a first series of DNA constructs encoding afirst and second chimeric protein and a second DNA construct encoding atarget gene under the transcriptional control of an transcriptioncontrol element responsive to the oligomerization of the chimericprotein molecules. The DNA construct encoding the first chimeric proteincomprises (a) at least one first receptor domain, capable of binding toa selected first ligand moiety, fused to (b) a heterologous additionalprotein domain capable of initiating a biological process upon [exposureto the oligomerization ligand, i.e. upon oligomerization of the firstchimeric protein to a second chimeric protein molecule. The DNAconstruct encoding the second chimeric protein comprises (i) at leastone receptor domain, capable of binding to a selected second ligandmoiety, fused to (ii) a heterologous additional protein domain capableof initiating a biological process upon exposure to the oligomerizationligand, i.e., upon oligomerization to the first chimeric protein. Thefirst and second receptor moieties in such cases may be the same ordifferent and the first and second selected ligand moieties may likewisebe the same or different.

[0030] DNA constructs encoding primary and/or optional chimeric proteinsof this invention may contain cell type specific transcriptionalregulatory elements. Such elements provide for tissue-specificexpression of the chimeras and thus for tissue-specific biologicalactivity triggered by their multimerization. To create transgenicanimals containing modified cells of this invention one may transfectthe desired constructs into ES cells, if they are available, ormicroinject the desired constructs directly into early embryos. See e.g.Watson et al, RECOMBINANT DNA (2d ed. 1992), esp. Chaps 14 and 24. Inthe latter case, use of a tissue-specific expression control sequence(promoter/enhancer) in the primary construct permits tissue-specificexpression of the primary chimeric protein(s). That in turn permitstissue-specific, ligand-regulatable triggering of apoptosis, i.e.,ligand-inducible ablation of cells in a tissue-specific manner. Byincorporation of a cell-type specific transcriptional regulatory elementin the optional chimera (in the presence or absence of constructs forthe primary chimera), ligand-regulated transcription of a target gene ortriggering of other biological events can likewise be achieved in atissue-specific manner. Animals and their progeny may be convenientlycharacterized by conventional genetic analysis. It should also be notedthat in addition to introduction into ES cells or early embryos, theconstructs may also be introduced by administration, e.g. in suitablevehicles or vectors, directly into the desired tissue of the wholeorganisms.

[0031] Our ligands are molecules capable of binding to two or morechimeric protein molecules of this invention to form an oligomerthereof, and have the formula:

[0032] linker-{rbm₁, rbm₂, . . . rbm_(n)}

[0033] wherein n is an integer from 2 to about 5, rbm₍₁₎₋rbm_((n)) arereceptor binding moieties which may be the same or different and whichare capable of binding to the chimeric protein(s). The rbm moieties arecovalently attached to a linker moiety which is a bi- ormulti-functional molecule capable of being covalently linked (“-”) totwo or more rbm moieties. Preferably the ligand has a molecular weightof less than about 5 kDa and is not a protein. Examples of such ligandsinclude those in which the rbm moieties are the same or different andcomprise an FK506-type moiety, a cyclosporin-type moiety, a steroid ortetracycline. Cyclosporin-type moieties include cyclosporin andderivatives thereof which are capable of binding to a cyclophilin,naturally occurring or modified, preferably with a Kd value below about10⁻⁶ M. In some embodiments it is preferred that the ligand bind to anaturally occurring receptor with a Kd value greater than about 10⁻⁶Mand more preferably greater than about 10⁵ M. Illustrative ligands ofthis invention are those in which at least one rbm comprises a moleculeof FK506, FK520, rapamycin or a derivative thereof modified at C9, C10or both, which ligands bind to a modified receptor or chimeric moleculecontaining a modified receptor domain with a Kd value at least one, andpreferably 2, and more preferably 3 and even more preferably 4 or 5 ormore orders of magnitude less than their Kd values with respect to anaturally occurring receptor protein. Linker moieties are also describedin detail later, but for the sake of illustration, include such moietiesas a C2-C20 alkylene, C4-C18 azalkylene, C6-C24 N-alkylene azalkylene,C6-C18 arylene, C8-C24 ardialkylene or C8-C36 bis-carboxamido alkylenemoiety.

[0034] The monomeric rbm's of this invention, as well as compoundscontaining sole copies of an rbm, which are capable of binding to ourchimeric proteins but not effecting dimerization or higher orderoligomerization thereof (in view of the monomeric nature of theindividual rbm) are oligomerization antagonists.

[0035] In an important aspect of ties invention, genetically engineeredcells of this invention can be grown together with other cells and canbe selectively ablated from the mixture of cells by addition of aneffective amount of an oligomerization ligand which corresponds to (i.e.is capable of binding to) the primary chimeric protein. Thus, contactingthe cells with the oligomerization ligand triggers cell death in theengineered cells. For example, such cells may be permitted to produce anendogenous or heterologous product for some desired period, and may thenbe deleted by addition of the ligand. In such cases, the cells areengineered to produce a primary chimera in accordance with thisinvention. The cells, which may be further engineered to express adesired gene under ligand-induced regulation, may be grown in culture byconventional means. In that case, addition to the culture medium of theligand for the optional chimera leads to expression of the desired geneand production of the desired protein. Expression of the gene andproduction of the protein can then be turned off by adding to the mediuman oligomerization antagonist reagent, as is described in detail below.In other cases, production of the protein is constitutive. In any event,the engineered cells can then be eliminated from the cell culture afterthey have served their intended purpose (e.g. production of a desiredprotein or other product) by adding to the medium an effective amount ofthe appropriate oligomerizing ligand to cause oligomerization of theprimary chimera and induce apoptosis in the engineered cells. Engineeredcells of this invention can also be used in vivo, to modify wholeorganisms, preferably animals, including humans, e.g. such that thecells produce a desired protein or other result within the animalcontaining such cells. Such uses include gene therapy. Alternatively,the chimeric proteins and oligomerizing molecules can be usedextracellularly to bring together proteins which act in concert toinitiate a physiological action.

[0036] This invention thus provides materials and methods forselectively ablating cells in response to the addition of anoligomerizing ligand. The method involves providing cells engineered inaccordance with this invention and exposing the cells to the ligand.

[0037] Thus, this invention provides a method for using cells engineeredas described herein for producing a heterologous protein via regulatedactivation of transcription of a target gene in the cells and foreliminating the engineered cells when desired. The method involvesproviding cells of this invention which express a primary chimericprotein capable, upon oligomerization, of initiating apoptosis, andwhich cells further contain and are capable of expressing (a) at leastone DNA construct encoding a chimeric protein capable, followingoligomerization, of activating transcription of (b) a target gene. Thechimeric protein comprises at least one receptor domain capable ofbinding to a selected oligomerization ligand. The receptor domain isfused to an action domain capable—on exposure to the oligomerizingligand, i.e., upon oligomerization with one or more other chimericproteins containing another copy of the action domain—of initiating anintracellular signal. That signal is capable of activating transcriptionof a gene, such as the target gene in this case, which is under thetranscriptional control of a transcriptional control element responsiveto that signal. The method thus involves exposing the cells to anoligomerization ligand capable of binding to the chimeric protein in anamount effective to result in expression of the target gene. In cases inwhich the cells are growing in culture, exposing them to the ligand iseffected by adding the ligand to the culture medium. In cases in whichthe cells are present within a host organism, exposing them to theligand is effected by administering the ligand to the host organism. Forinstance, in cases in which the host organism is an animal, inparticular, a mammal (including a human), the ligand is administered tothe host animal by oral, bucal, sublingual, transdermal, subcutaneous,intramuscular, intravenous, intra-joint or inhalation administration inan appropriate vehicle therefor. To ablate the engineered cells, oneadds to the culture medium or administers to the host organism, as thecase may be, a second oligomerizing ligand which is capable ofoligomerizing the primary chimera.

[0038] This invention further encompasses pharmaceutical compositionsfor eliminating genetically engineered cells of this invention from amixture of different cells, including from animal tissue or from asubject containing such engineered cells. Such pharmaceuticalcompositions comprise an oligomerization ligand of this invention inadmixture with a pharmaceutically acceptable carrier and optionally withone or more pharmaceutically acceptable excipients. The oligomerizationligand can be a homo-oligomerization reagent or a hetero-oligomerizationreagent as described in detail elsewhere so long as it is capable ofbinding to a primary chimeric protein of this invention or triggeringapoptosis of engineered cells of this invention. Likewise, thisinvention further encompasses a pharmaceutical composition comprising anoligomerization antagonist of this invention admixture with apharmaceutically acceptable carrier and optionally with one or morepharmaceutically acceptable excipients for preventing or reducing, inwhole or part, the level of oligomerization of chimeric proteins inengineered cells of this invention, in cell culture or in a subject, andthus for preventing or de-activating cell death in the relevant cells.Thus, the use of the oligomerization reagents and of the oligomerizationantagonist reagents to prepare pharmaceutical compositions isencompassed by this invention.

[0039] This invention also offers a method for providing a hostorganism, preferably an animal, and in many cases a mammal, responsiveto an oligomerization ligand of his invention. The method involvesintroducing into the organism cells which have been engineered ex vivoin accordance with this invention, i.e. containing a DNA constructencoding a chimeric protein hereof, and so forth. Alternatively, one canintroduce the DNA constructs of this invention into a host organism,e.g. mammal under conditions permitting transfection of one or morecells of the host mammal in vivo.

[0040] We further provide kits for producing cells susceptable toligand-regulated apoptosis. One kit contains at least one DNA constructencoding one of our primary chimeric proteins, containing at least onereceptor domain and an action domain (e.g., the cytoplasmic domain ofFas or of a TNF receptor, as described elsewhere). In one embodiment theDNA constrict contains a conventional polylinker to provide thepractitioner a site for the incorporation of cell-type specificexpression control element(s) (promoter and/or enhancer elements) toprovide for cell-type or tissue-specific expression of one or more ofthe chimeras. The kit may contain a quantity of a ligand of thisinvention capable of oligomerizing the chimeric protein moleculesencoded by the DNA constructs of the kit, and may contain in addition aquantity of an oligomerization antagonist e.g. monomeric ligand reagent.Where a sole chimeric protein is encoded by the construct(s), theoligomerization ligand is a homo-oligomerization ligand. Where more thanone such chimeric protein is encoded, a hetero-oligomerization ligandmay be included. The kit may further contain a additional DNA constructsencoding optional chimera and/or target gene constructs and/or atranscription control element responsive to oligomerization of thechimeric protein molecules. The DNA constructs will preferably beassociated with one or more selection markers for convenient selectionof transfectants, as well as other conventional vector elements usefulfor replication in prokaryotes, for expression in eukaryotes, and thelike. The selection markers may be the same or different for eachdifferent DNA construct, permitting the selection of cells which containvarious combinations of such DNA construct(s).

[0041] For example, one kit of this invention contains a DNA constructencoding a primary chimeric protein as described elsewhere; a firstoptional DNA construct encoding a chimeric protein containing at listone receptor domain (capable of binding to a selected ligand), fused toa transcriptional activator domain; a second optional DNA constructencoding a chimeric protein containing at least one receptor domain(capable of binding to a selected ligand), fused to a DNA bindingdomain; and a target gene DNA construct encoding a target gene under thecontrol of a transcriptional control element containing a DNA sequenceto which the DNA binding domain binds and which is transcriptionallyactivated by exposure to the ligand in the presence of the first andsecond optional chimeric proteins.

[0042] Alternatively, a DNA construct for introducing a target geneunder the control of a responsive transcriptional control element maycontain a cloning site in place of a target gene to provide a kit forengineering cells to inducably express a gene to be provided by thepractitioner.

[0043] Other kits of this invention may contain one or two (or more) DNAconstructs for chimeric proteins in which one or more contain a cloningsite in place of an action domain (transcriptional initiation signalgenerator, transcriptional activator, DNA binding protein, etc.),permitting the user to insert whichever action domain s/he wishes. Sucha kit may optionally include other elements as described above, e.g. DNAconstruct for a target gene under responsive expression control,oligomerization ligand, antagonist, etc.

[0044] Any of the kits may also contain positive control cells whichwere stably transformed with constructs of this invention such that theyexpress a reporter gene (for CAT, beta-galactosidase or any convenientlydetectable gene product) in response to exposure of the cells to theligand. Reagents for detecting and/or quantifying the expression of thereporter gene may also be provided.

BRIEF DESCRIPTION OF THE FIGURES

[0045]FIG. 1 is a diagram of the plasmid pSXNeo/IL2 (IL2-SX). InNF-AT-SX, the HindIII-ClaI DNA fragment from IL2-SX containing the IL2enhancer/promoter, is replaced by a minimal IL-2 promoter conferringbasal transition and an inducible element containing three tandemNFAT-binding sites (described below).

[0046]FIG. 2 is a flow diagram of the preparation of the intracellularsignaling chimera plasmids and p#MXFn and p#MFnZ, where n indicates thenumber of binding domains.

[0047]FIGS. 3A and 3B are a flow diagram of the preparation of theextracellular signalling chimera plasmid p#1FK3/pBJ5.

[0048]FIGS. 4A, 4B and 4C are sequences of the primers use in theconstructions of the plasmids employed in the subject invention.

[0049]FIG. 5 is a chart of the response of reporter constructs havingdifferent enhancer groups to reaction of the receptor TAC/CD3 ζ with aligand.

[0050]FIG. 6 is a chart of the activity of various ligands with the TAgJurkat cells described in Example 1. For FIG. 6B, see also Spencer etal, Science 262, 1019, FIG. 3 and capation, esp. 3B on p. 1020 therein.

[0051]FIG. 7 is a chart of the activity of the ligand FK1012A (8 FIG.9B) with the extracellular receptor 1FK3 (FKBPx3/CD3 ζ.

[0052]FIG. 8 is a chart of the activation of an NFAT reporter viasignalling through a myristoylated CD3 ζ/FKBP12 chimera.

[0053]FIGS. 9A and 9B are the chemical structures of the allyl-linkedFK506 variants and the cyclohexyl-linked FK506 variants, respectively.

[0054]FIG. 10 is a flow diagram of the synthesis of derivatives ofFK520.

[0055]FIGS. 11A and B are a flow diagram of a synthesis of derivativesof FK520 and chemical structures of FK520, where the bottom structuresare designed to bind to mutant FKBP12.

[0056]FIG. 12 is a diagrammatic depiction of mutant FKBP with a modifiedFK520 in the putative deft.

[0057]FIG. 13 is a flow diagram of the synthesis of heterodimers ofFK520 and cyclosporin.

[0058]FIG. 14 is a schematic representation of the oligomerization ofchimeric proteins, illustrated by chimeric proteins containing animmunophilin moiety as the receptor domain.

[0059]FIG. 15 depicts ligand-mediated oligomerization of chimericproteins, showing schematically the triggering of a transcriptionalinitiation signal.

[0060]FIG. 16 depicts synthetic schemes for HED and HOD reagents basedon FK506-type moieties.

[0061]FIG. 17 depicts the synthesis of (CsA)2 beginning with CsA.

[0062]FIG. 18 is an overview of the fusion cDNA construct and proteinMZF3E.

[0063]FIG. 19 shows FK1012-induced cell death of the Jurkat T-cell linetransfected with a myristoylated Fas-FKBP12 fusion protein (MFF3E), asindicated by the decreased transcriptional activity of the cells.

[0064]FIG. 20A is an analysis of cyclophilin-Fas (and Fas-cyclophilin)fusion constructs in the transient transfection assay. MC3FE was shownto be the most effective in this series.

[0065]FIG. 20B depicts Immunophilin-Fas antigen chimeras and results oftransient expression experiments in Jurkat T cells stably transformedwith large T-antigen. Myr: the myristylaion sequence taken frompp60^(c-src) encoding residues 1-14 (Wilson et al. Mol & Cell Biol 9 4(1989): 1536-44); FKBP: human FKBP12; CypC: murine cyclophilin Csequence encoding residues 36-212 (Freidman et al, Cell 66 4 (1991):799-806); Fas: intracellular domain of human Fas antigen encodingresidues 179-319 (Oehm et al, J Biol Chem 267 15 (1992): 10709-15).Cells were electroporated with a plasmid encoding a secreted alkalinephosphatase reporter gene under the control of 3 tandem AP1 promotersalong with a six fold molar excess of the immunophilin fusion construct.After 24 h the cells were stimulated with PMA (50 ng/mL), whichstimulates the synthesis of the reporter gene, and (CsA)2. At 48 h thecells were assayed for reporter gene activity. Western blots wereperformed at 24 h using anti-HA epitope antibodies.

[0066]FIG. 21 depicts the synthesis of modified FK506 type compounds.

DESCRIPTION

[0067] I. General Discussion

[0068] This invention provides chimeric proteins, organic molecules foroligomerizing the chimeric proteins and a system for using them. Thefused proteins (chimeras) have a binding domain for binding to the(preferably small) organic oligomerizing molecule and an action domain,which can effectuate a physiological action or cellular process as aresult of oligomerization of the chimeric proteins.

[0069] The basic concept for inducible protein association isillustrated in FIG. 14. Ligands which can function as heterodimerization(or hetero-oligomerization, “HED”) and homodimerization (orhomo-oligomerization, “HOD”) agents are depicted as dumbell-shapedstructures.

[0070] (Homodimerization and homo-oligomerization refer to theassociation of like components to form dimers or oligomers, linked asthey are by the ligands of this invention. Heterodimerization andhetero-oligomerization refer to the association of dissimilar componentsto form dimers or oligomers. Homo-oligomers thus comprise an associationof multiple copies of a particular component while hetero-oligomerscomprise an association of copies of different components.“Oligomerization”, “oligomerize” and “oligomer”, as the terms are usedherein, with or without prefixes, are intended to encompass“dimerization”, “dimerize” and “dimer”, absent an explicit indication tothe contrary.)

[0071] Also depicted in FIG. 14 are fusion protein molecules containinga target protein domain of interest (“action domain”) and one or morereceptor domains that can bind to the ligands. For intracellularchimeric proteins, i.e., proteins which are located within the cells inwhich they are produced, a cellular targeting sequence (includingorganelle targeting amino acid sequences) will preferably also bepresent. Binding of the ligand to the receptor domains hetero- orhomodimerizes the fusion proteins. Oligomerization brings the actiondomains into close proximity with one another thus triggering cellularprocesses normally associated with the respective action domain—such asapotosis or TCR-mediated signal transduction, for example.

[0072] Cellular processes which can be triggered by oligomerizationinclude a change in state, such as a physical state, e.g. conformationalchange, change in binding partner, cell death, initiation oftranscription, channel opening, ion release, e.g. Ca⁺² etc. or achemical state, such as an enzymatically catalyzed chemical reaction,e.g. acylation, methylation, hydrolysis, phosphorylation ordephosphorylation, change in redox state, rearrangement, or the like.Thus, any such process which can be triggered by ligand-mediatedoligomerization is included within the scope of this invention, althougha primary focus here is apoptosis.

[0073] In a central feature of this invention, cells are modified so asto be responsive to ligand molecules which are capable of binding to,and thus oligomerizing the primary chimeras disclosed herein. See e.g.Examples 4(B) and 4(C), infra. Such engineered cells respond to thepresence of ligand by undergoing apoptosis and may thus be eliminated inapplications of gene therapy and other situations where it is necessaryor desirable to ablate the genetically modified cells. For example, themodified cells may become cancerous or otherwise deleterious orsuperfluous.

[0074] The modified cells are characterized by a genome containing agenetic construct (or series thereof encoding a primary chimeric proteinof this invention, which permits ligand-regulated apoptosis. The primarychimera contains, e.g., the cytoplasmic domain of the fas antigen orApo-1 antigen, which when cross-liked, induces apoptosis in most celltypes (Trauth et al. (1989) Science 245, 301-305; Watanaba-Fukunaga etal. (1992) Nature 356, 314). In this way one can provide forligand-inducable cell death for an engineered population of cells.

[0075] The cells may be further engineered to produce optionaladditional chimeric proteins capable of binding with, and beingresponsive to, selected ligand molecules. Such further optionalengineering imparts additional ligand-regulatable functionality on thecells, which can be used in applications involving in vitro cell cultureand in gene therapy applications. Preferably, the ligand molecules whichare capable of binding to the optional additional chimera(s) andregulating the optional additional cellular processes (such as genetranscription, for example) do not cross react with the primary chimeramolecules, and therefore do not trigger apoptosis in the engineeredcells.

[0076] Such further modified cells can be used in applications in whichregulation of cellular processes such as transcription or translation(both are included under the term expression) of a target gene isdesired. Such cells are characterized by a genome containing at least afirst or first series (the series may include only one construct) ofgenetic constructs encoding the optional additional chimeras, anddesirably a second or second series (the series may include only oneconstruct) of target gene constructs.

[0077] The nature and number of such genetic constructs will depend onthe nature of the chimeric protein and the role it plays in the cell.For instance, in embodiments where the optional additional chimericprotein is to be associated with expression of a target gene (and whichmay contain an intracellular targeting sequence or domain which directsthe chimeric protein to be associated with the cellular surface membraneor with an organelle e.g. nucleus or vesicle), then there will normallybe at least two series of such additional constructs: a first seriesencoding the chimeric protein(s) which upon ligand-mediatedoligomerization initiate a signal directing target gene expression, anddesirably a second series which comprise the target gene and/orexpression control elements therefor which are responsive to the signal.

[0078] Only a single construct in the first series will be requiredwhere a homooligomer, usually a homodimer, is involved, while two ormore, usually not more than three constructs may be involved, where aheterooligomer is involved. The chimeric proteins encoded by the firstseries of constructs will be associated with actuation of genetranscription and will normally be directed to the surface membrane orthe nucleus, where the oligomerized chimeric protein is able toinitiate, directly or indirectly, the transcription of one or moretarget genes. A second series of additional constructs will be requiredwhere an exogenous gene(s) is introduced, or where an exogenous orrecombinant expression control sequence is introduced (e.g. byhomologous recombination) for expression of an endogenous gene, ineither case, whose transcription will be activated by the oligomerizingof the chimeric protein.

[0079] A different first series of additional constructs is employedwhere the chimeric proteins are intracellular and can act directlywithout initiation of transcription of another gene. For example,proteins associated with exocytosis can be expressed inducibly orconstitutively, where the proteins will not normally complex except inthe presence of the oligomerizing molecule. By employing proteins whichhave any or all of these properties which do not complex in the hostcell; are inhibited by complexation with other proteins, whichinhibition may be overcome by oligomerization with the ligand; requireactivation through a process which is not available in the host cell, orby modifying the proteins which direct fusion of a vesicle with theplasma membrane to form chimeric proteins, where the extent of complexformation and membrane fusion is enhanced in the presence of theoligomerizing molecule, exocytosis is or has the ability to be inducedby the oligomerizing molecule.

[0080] Other intracellular proteins, such as kinases, phosphatases andcell cycle control proteins can be similarly modified and used.

[0081] Various classes of optional additional genetic constructs usefulin the practice of is invention are described as follows:

[0082] (1) constructs which encode a chimeric protein comprising abinding domain and an action domain, where the binding domain isextracellular or intracellular and the action domain is intracellularsuch that ligand-mediated oligomerization of the chimeric protein byitself (to form a homo-oligomer) or with a different fused proteincomprising a different action domain (to form a hetero-oligomer),induces a signal which results in a series of events resulting intranscriptional activation of one or more genes;

[0083] (2) constructs which encode a chimeric protein having a bindingdomain and an action domain, where the binding domain and action domainare in the nucleus, such that ligand-mediated oligomerization of theprotein, by itself (to form a homo-oligomer) or with a different fusedprotein comprising a different action domain (to form a heterooligomer),induces initiation of transcription directly via complexation of theoligomer(s) with the DNA transcriptional initiation region;

[0084] (3) constructs which encode a chimeric protein containing abinding domain and an action domain, where the binding domain and theaction domain are cytoplasmic, such that ligand-mediated oligomerizationof the protein, by itself (to form a homo-oligomer) or with a differentfused protein comprising a different action domain (to form ahetero-oligomer), results in exocytosis; and

[0085] (4) constructs which encode a chimeric protein containing abinding domain and an action domain, where the binding domain and actiondomain are extracellular and the action domain is associated withinitiating a biological activity (by way of non-limiting illustration,the action domain can itself bind to a substance, receptor or othermembrane protein yielding, upon ligand-mediated oligomerization of thechimeras, the bridging of one or more similar or dissimilar molecules orcells); and,

[0086] (5) constructs which encode a destabilizing, inactivating orshort-lived chimeric protein having a binding domain and an actiondomain, such that ligand-mediated oligomerization of the protein with atarget protein comprising a different action domain leads to thedestabilization and/or degradation or inactivation of said oligomerizedtarget protein.

[0087] II. Transcription Regulation

[0088] The construct(s) of Groups (1) and (2), above, will be consideredfirst. Group (1) constructs differ from group (2) constructs in theireffect Group (1) constructs are somewhat pleiotropic, i.e. capable ofactivating a number of wild-type genes, as well as the target gene(s).In addition, the response of the expression products of group (1) genesto the ligand is relatively slow. Group (2) constructs can be directedto a specific target gene and are capable of limiting the number ofgenes which will be transcribed. The response of expression products ofgroup (2) constructs to the ligand is very rapid.

[0089] The subject system for groups (1) and (2) will include a firstseries of constructs which comprise DNA sequences encoding the chimericproteins, usually involving from one to three, usually one to two,different constructs. The system usually will also include a secondseries of constructs which will provide for expression of one or moregenes, usually an exogenous gene. By “exogenous gene” is meant a genewhich is not otherwise normally expressed by the cell, e.g. because ofthe nature of the cell, because of a genetic defect of the cell, becausethe gene is from a different species or is a mutated or synthetic gene,or the like. Such gene can encode a protein, antisense molecule,ribozyme etc., or can be a DNA sequence comprising an expression controlsequence linked or to be linked to an endogenous gene with which theexpression control sequence is not normally associated. Thus, asmentioned before, the construct can contain an exogenous or recombinantexpression control sequence for ligand-induced expression of anendogenous gene.

[0090] The chimeric protein encoded by a construct of groups (1), (2)and (3) can have, as is often preferred, an intracellular targetingdomain comprising a sequence which directs the chimeric protein to thedesired compartment, e.g. surface membrane, nucleus, vesicular membrane,or other site, where a desired physiological activity can be initiatedby the ligand-mediated oligomerization, at least dimerization, of thechimeric protein.

[0091] The chimeric protein contains a second (“binding” or “receptor”)domain which is capable of binding to at least one ligand molecule.Since the ligand can contain more than one binding site or epitope, itcan form dimers or higher order homo- or hetero-oligomers with thechimeric proteins of this invention. The binding domain of the chimericprotein can have one or a plurality of binding sites, so thathomooligomers can be formed with a divalent ligand. In this way theligand can oligomerize the chimeric protein by having two or moreepitopes to which the second domain can bind, thus providing for higherorder oligomerization of the chimeric protein.

[0092] The chimeric protein also contains a third (“action”) domaincapable of initiating a biological activity upon ligand-mediatedoligomerization of chimeric protein molecules via the binding domains.Thus, the action domain may be associated with transduction of a signalas a result of the ligand-mediated oligomerization. Such signal, forinstance, could result in the initiation of transcription of one or moregenes, depending on the particular intermediate components involved inthe signal transduction. See FIG. 15 which depicts an illustrativechimeric protein in which the intracellular targeting domain comprises amyristate moiety; the receptor domain comprises three FKBP12 moieties;and the action domain comprises a zeta subunit. In other chimericproteins the action domains may comprise transcription factors, whichupon oligomerization, result in the initiation of transcription of oneor more target genes, endogenous and/or exogenous. The action domainscan comprise proteins or portions thereof which are associated withfusion of vesicle membranes with the surface or other membrane, e.g.proteins of the SNAP and SNARE groups (See, Sollner et al., Nature(1993) 362, 318 and 353; Cell (1993) 72, 43).

[0093] A. Surface Membrane Receptor

[0094] One class of additional optional chimeric proteins of thisinvention are involved with the surface membrane and are capable oftransducing a signal leading to the transcription of one or more genes.The process involves a number of auxiliary proteins in a series ofinteractions culminating in the binding of transcription factors topromoter regions associated with the target gene(s). In cases in whichthe transcription factors bind to promoter regions associated with othergenes, transcription is initiated there as well. A construct encoding achimeric protein of this embodiment can encode a signal sequence whichcan be subject to processing and therefore may not be present in themature chimeric protein. The chimeric protein will in any event comprise(a) a binding domain capable of binding a predetermined ligand, (b) anoptional (although in many embodiments, preferred) membrane bindingdomain which includes a transmembrane domain or an attached lipid fortranslocating the fused protein to the cell surface/membrane andretaining the protein bound to the cell surface membrane, and, (c) asthe action domain, a cytoplasmic signal initiation domain. Thecytoplasmic signal initiation domain is capable of initiating a signalwhich results in transcription of a gene having a recognition sequencefor the initiated signal in the transcriptional initiation region.

[0095] The gene whose expression is regulated by the signal from thechimeric protein is referred to herein as the “target” gene, whether itis an exogenous gene or an endogenous gene under the expression controlof an endogenous or exogenous (or hybrid) expression control sequence.The molecular portion of the chimeric protein which provides for bindingto a membrane is also referred to as the “retention domain”. Suitableretention domains include a moiety which binds directly to the lipidlayer of the membrane, such as through lipid participation in themembrane or extending through the membrane, or the like. In such casesthe protein becomes translocated to and bound to the membrane,particularly the cellular membrane, as depicted in FIG. 15.

[0096] B. Nuclear Transcription Factors

[0097] Another optional first construct encodes a chimeric proteincontaining a cellular targeting sequence which provides for the proteinto be translocated to the nucleus. This (“signal consensus”) sequencehas a plurality of basic amino acids, referred to as a bipartite basicrepeat (reviewed in Garcia-Bustos et al, Biochimica et Biophysica Acta(1991) 1071, 83-101). This sequence can appear in any portion of themolecule internal or proximal to the N- or C-terminus and results in thechimeric protein being inside the nucleus. The practice of oneembodiment of this invention will involve at least two (“first series”)chimeric proteins: (1) one having an action domain which binds to theDNA of the transcription initiation region associated with a target geneand (2) a different chimeric protein containing as an action domain, atranscriptional activation domain capable, in association with the DNAbinding domain of the first chimeric protein, of initiatingtranscription of a target gene. The two action domains or transcriptionfactors can be derived from the same or different protein molecules.

[0098] The transcription factors can be endogenous or exogenous to thecellular host. If the transcription factors are exogenous, butfunctional within the host and can cooperate with the endogenous RNApolymerase (rather than requiring an exogenous RNA polymerase, for whicha gene could be introduced), then an exogenous promoter elementfunctional with the fused transcription factors can be provided with asecond construct for regulating transcription of the target gene. Bythis means the initiation of transcription can be restricted to thegene(s) associated with the exogenous promoter region, i.e., the targetgene(s).

[0099] A large number of transcription factors are known which requiretwo subunits for activity. Alternatively, in cases where a singletranscription factor can be divided into two separate functional domains(e.g. a transcriptional activator domain and a DNA-binding domain), sothat each domain is inactive by itself, but when brought together inclose proximity, transcriptional activity is restored. Transcriptionfactors which can be used include yeast GAL4, which can be divided intotwo domains as described by Fields and Song, supra. The authors use afusion of GAL4(1-147)-SNF1 and SNF4GAL4(768-881), where the SNF1 and -4may be replaced by the subject binding proteins as binding domains.Combinations of GAL4 and VP16 or HNF-1 can be employed. Othertranscription factors are members of the Jun, Fos, and ATF/CREBfamilies, Oct1, Sp1, HNF-3, the steroid receptor superfamily, and thelike.

[0100] As an alternative to using the combination of a DNA bindingdomain and a naturally occurring activation domain or modified formthereof, the activation domain may be replaced by one of the bindingproteins associated with bridging between a transcriptional activationdomain and an RNA polymerase, including but not limited to RNApolymerase II. These proteins include the proteins referred to as TAFs,the TFII proteins, particularly B and D, or the like. Thus, one can useany one or combination of proteins, for example, fused proteins orbinding motifs thereof, which serve in the bridge between the DNAbinding protein and RNA polymerase and provide for initiation oftranscription. Preferably, the protein closest to the RNA polymerasewill be employed in conjunction with the DNA binding domain to providefor initiation of transcription. If desired, the subject constructs canprovide for three or more, usually not more than about 4, proteins to bebrought together to provide the transcription initiation complex.

[0101] Rather than have a transcriptional activation domain as an actiondomain, an inactivation domain, such as ssn-6/TUP-1 or Krüppel-familysuppressor domain can be employed. In this manner, regulation results inturning off the transcription of a gene which is constitutivelyexpressed. For example, in the case of gene therapy one can provide forconstitutive expression of a hormone, such as growth hormone, bloodproteins, immunoglobulins, etc. By employing constructs encoding onechimeric protein containing a DNA binding domain joined to a ligandbinding domain and another chimeric protein containing an inactivationdomain joined to a ligand binding domain, the expression of the gene canbe inhibited via ligand-mediated oligomerization.

[0102] Constructs encoding a chimeric protein containing inter alia aligand-binding domain fused to a transcriptional activating domain orsubunit, transcriptional inactivating domain or DNA-binding domain aredesigned and assembled in the same manner as described for the otherconstructs. Frequently, the N-terminus of the transcription factor willbe bound to the C-terminus of the ligand-binding domain, although insome cases the reverse will be true, for example, where two individualdomains of a single transcription factor are divided between twodifferent chimeras.

[0103] III. Exocytosis

[0104] Another application of the ligand-mediated oligomerizationmechanism is exocytosis, where export of a protein rather thantranscription is controlled by the ligand. This can be used inconjunction with the expression of one or more proteins of interest, asan alternative to providing for secretion of the protein(s) of interestvia a secretory signal sequence. This embodiment involves two differentfirst constructs. One construct encodes a chimeric protein which directsthe protein to the vesicle to be integrated into the vesicular membraneas described by Sollner et al., supra. Proteins which may be used as thevesicle binding protein include VAMP (synaptobrevin), SNC2, rab3, SEC4,synaptotagmin, e, individually or in combination. The cellular membraneprotein may include syntaxin, SSO1, SSO2, neurexin, etc., individuallyor in combination. The other construct provides for transport to thesurface membrane and employs the myristoyl signal sequence, other plasmamembrane targeting sequence (e.g. for prenylation) or transmembraneretention domain, as described above. The encoded proteins are describedin the above references and, all or functional part, may serve as theaction domains. These constructs could be used in conjunction with theexpression of an exogenous protein, properly encoded for transport to avesicle or for an endocytotic endogenous protein, to enhance export ofthe endogenous protein.

[0105] Various mechanisms can be employed for exocytosis. Depending onthe cell type and which protein is limiting for endocytosis in the cell,one or more of the vesicle bound proteins or cellular proteins may beencoded by one or more constructs having a response element which isactivated by the ligand. Of particular interest is the combination ofVAMP and syntaxin. Alternatively, one can provide for constitutiveexpression of non-limiting proteins controlling exocytosis and providefor ligand regulated expression of the exocytosis limiting protein.Finally, one can provide for constitutive expression of the chimericproteins associated with exocytosis, so that exocytosis is controlled byoligomerizing the chimeric proteins with the ligand. By employingappropriate binding domains, one can provide for different chimericproteins to be oligomerized on the vesicle surface to form an activecomplex, and/or linking of the vesicle protein(s) with the cell membranesurface protein through the ligand. The chimeric proteins may notprovide for exocytosis in the absence of the ligand due to modificationsin the ligand which substantially reduce the binding affinity betweenthe proteins governing exoytosis, such as deletions, mutations, etc.These modifications can be readily determined by employing overlappingfragments of the individual proteins and determining which fragmentsretain activity. The fragments can be further modified by using alaninesubstitutions to determine the individual amino acids whichsubstantially affect binding (Beohncke et al., J. Immunol. (1993) 150,331-341; Evavold et al., ibid (1992) 148, 347-353).

[0106] The proteins assembled in the lumen of the vesicle, as well asthe fused proteins associated with exocytosis can be expressedconstitutively or inducibly, as described above. Depending on thepurpose of the exocytosis, whether endogenous or exogenous proteins areinvolved, whether the proteins to be exported are expressedconstitutively or inducibly, whether the same ligand can be used forinitiating transcription of the fused proteins associated withexocytosis and the proteins to be exported, or whether the differentproteins are to be subject to different inducible signals, may determinethe manner in which expression is controlled. In one aspect, theexocytosis mechanism would be the only event controlled by the ligand.In other aspects, both expression of at least one protein and exocytosismay be subject to ligand control.

[0107] Various proteins may be modified by introduction of a cellulartargeting sequence for translocation of the protein to a vesicle withoutloss of the physiological activity of the protein. By using exocytosisas the delivery mechanism relatively high dosages may be deliveredwithin a short period of time to produce a high localized level of theprotein or a high concentration in the vascular system, depending on thenature of the host. Proteins of interest include e.g. insulin, tissueplasminogen activator, cytokines, erythropoietin, colony stimulatingfactors, growth factors, inflammatory peptides, cell migration factors.

[0108] Coding sequences for directing proteins to a vesicle areavailable from the vesicle binding proteins associated with exocytosis.See, for example, Sollner, et al. supra.

[0109] Another use of the oligomerization mechanism is the control ofprotein degradation or inactivation. For example, a relatively stable orlong-lived chimeric protein of this invention can be destabilized ortargeted for degradation by ligand-mediated oligomerization with adifferent chimeric protein of this invention which has a relativelyshort half-life or which otherwise destabilizes or targets the oligomerfor degradation. In this embodiment, ligand-mediated oligomerizationregulates biological functioning of a protein by conferring upon it intrans a shortened half-life. The latter chimeric protein may contain adomain targeting the protein to the lysosome or a domain rendering theprotein susceptible to proteolytic cleavage in the cytosol or nucleus ornon-lysosomal organelle.

[0110] The half-life of proteins within cells is determined by a numberof factors which include the presence of short amino acid sequenceswithin said protein rich in the amino acid residues proline, glutamicadd, serine and threonine, hence “PEST”, other sequences with similarfunction, protease sensitive cleavage sites and the state ofubiquitinization. Ubiquitinization is the modification of a protein byone or more units of the short polypeptide chains ubiquitin, whichtargets proteins for degradation. The rate of ubiquitinization ofproteins is considered to be determined primarily by the identity of theN-terminal amino acid of the processed protein and one or more uniquelysine residues near the amino terminus.

[0111] IV. Other Regulatory Systems

[0112] Other biological functions which can be controlled byoligomerization of particular activities associated with individualproteins are protein kinase or phosphatase activity, reductase activity,cyclooxygenase activity, protease activity or any other enzymaticreaction dependent on subunit association. Also, one may provide forassociation of G proteins with a receptor protein associated with thecell cycle, e.g. cyclins and cdc kinases, multiunit detoxifying enzymes.

[0113] V. Components of Constructs

[0114] The second or additional optional constructs (target geneconstructs) associated with group (1) and (2) optional additionalchimeric proteins comprise a transcriptional initiation region havingthe indicated target recognition sequence or responsive element, so asto be responsive to signal initiation from the activated receptor oractivated transcription factors resulting in at least one gene ofinterest being transcribed to a sequence(s) of interest, usually mRNA,whose transcription and, as appropriate, translation may result in theexpression of a protein and/or the regulation of other genes, e.g.antisense, expression of transcriptional factors, expression of membranefusion proteins, etc.

[0115] For the different purposes and different sites, different bindingdomains and different cytoplasmic domains will be used. For chimericprotein receptors associated with the surface membrane, if theligand-binding domain is extracellular, the chimeric protein can bedesigned to contain an extracellular domain selected from a variety ofsurface membrane proteins. Similarly, different cytoplasmic orintracellular domains of the surface membrane proteins which are able totransduce a signal can be employed, depending on which endogenous genesare regulated by the cytoplasmic portion. Where the chimeric protein isinternal, internal to the surface membrane protein or associated with anorganelle, e.g. nucleus, vesicle, etc., the ligand-binding domainprotein will be restricted to domains which can bind molecules which cancross the surface membrane or other membrane, as appropriate. Therefore,these binding domains will generally bind to small naturally occurringor synthetic ligand molecules which do not involve proteins or nucleicacids.

[0116] A. Cytoplasmic Domains

[0117] A chimeric protein receptor of Group (1) can contain acytoplasmic domain from one of the various cell surface membranereceptors or variants thereof (or other action domains for that matter)for which a corresponding recognition sequence is known or availablewhich is capable of initiating transcription in response tomultimerization of the chimeric proteins. Such recognition sequencesinclude those associated with a gene responsive to transcriptionalactivation triggered by such a receptor. Mutant receptors of interestwill dissociate transcriptional activation of a target gene fromactivation of genes which can be associated with harmful side effects,such as deregulated cell growth or inappropriate release of cytokines.The receptor-associated cytoplasmic domains of particular interest willhave the following characteristics: receptor activation leads toinitiation of transcription for relatively few (desirably fewer than100) and generally innocuous genes in the cellular host; the otherfactors necessary for transcription initiated by receptor activation arepresent in the cellular host; genes which are activated other than thetarget genes will not affect the intended purpose for which these cellsare to be used; oligomerization of the cytoplasmic domain or otheravailable mechanism results in signal initiation; and joining of thecytoplasmic domain to a desired ligand-binding domain will not interferewith signalling. A number of different cytoplasmic domains are known.Many of these domains are tyrosine kinases or are complexed withtyrosine kinases, e.g. CD3 ζ, IL-2R, IL-3R, etc. For a review seeCantley, et al., Cell (1991) 64, 281. Tyrosine kinase receptors whichare activated by crosslinking, e.g. dimerization (based on nomenclaturefirst proposed by Yarden and Ulrich, Annu. Rev. Biochem. (1988) 57, 443,include subclass I: EGF-R, ATR2/neu, HER2/neu, HER3/c-erbB-3, Xmrk;subclass II: insulin-R, IGF-1-R[insulin-like growth factor receptor],IRR; subclass III: PDGF-R-A, PDGF-R-B, CSF-1-R (M-CSF/c-Fms), c-kit,STK-1/Flk-2; and subclass IV: FGF-R, flg [acidic FGF], bek [basic FGF]);neurotrophic tryosine kinases: Trk family, includes NGF-R, Ror1,2.Receptors which associate with tyrosine kinases upon a cross-linkinginclude the CD3 ζ-family: CD3 ζ and CD3 1 η (found primarily in T cells,associates with Fyn); β and γ chains of Fc_(ε)RI (found primarily inmast cells and basophils); γ chain of Fc_(γ)RIII/CD16 (found primarilyin macrophages, neutrophils and natural killer cells); CD3 γ, -δ, and -ε(found primarily in T cells); Ig-α/MB-1 and Ig-β/B29 (found primarily inB cell). Many cytokine and growth factor receptors associate with commonβ subunits which interact with tyrosine kinases and/or other signallingmolecules and which can be used as cytoplasmic domains in chimericproteins of this invention. These include (1) the common β subunitshared by the GM-CSF, IL-3 and IL-5 receptors; (2) the β chain gp130subunit associated with the IL-6, leukemia inhibitory factor (LIF),ciliary neurotrophic factor (CNTF), oncostatin M, and IL-11 receptors;(3) the IL-2 receptor γ subunit associated also with receptors for IL-4,IL-7 and IL-13 (and possibly IL-9); and (4) the β chain of the IL-2receptor which is homologous to the cytoplasmic domain of the G-CSFreceptor.

[0118] The interferon family of receptors which include interferons α/βand γ (which can activate one or more members of the JAK, Tyk family oftyrosine kinases) as well as the receptors for growth hormone,erythropoietin and prolactin (which also can activate JAK2) can also beused as sources for cytoplasmic domains.

[0119] Other sources of cytoplasmic domains include the TGF-β family ofcell surface receptors (reviewed by Kingsley, D., Genes and Development1994 8 133). This family of receptors contains serin/threonine kinaseactivity in their cytoplasmic domains, which are believed to be actiatedby crosslinking.

[0120] The tyrosine kinases associated with activation and inactivationof transcription factors are of particular interest in providingspecific pathways which can be controlled and can be used to initiate orinhibit expression of an exogenous gene.

[0121] The following table provides a number of receptors andcharacteristics associated with the receptor and their nuclear responseelements that activate genes. The list is not exhaustive, but providesexemplary systems for use in the subject invention.

[0122] In many situations mutated cytoplasmic domains can be obtainedwhere the signal which is transduced may vary from the wild type,resulting in a restricted or different pathway as compared to thewild-type pathway(s). For example, in the case of growth factors, suchas EGF and FGF, mutations have been reported where the signal isuncoupled from cell growth but is still maintained with c-fos (Peters,et al., Nature (1992) 358, 678).

[0123] The tyrosine kinase receptors can be found on a wide variety ofcells throughout the body. In contrast, the CD3 ζ-family, the Ig familyand the lymphokine β-chain receptor family are found primarily onhematopoietic cells, particularly T-cells, B-cells, mast cells,basophils, macrophages, neutrophils, and natural killer cells. Thesignals required for NF-AT transcription come primarily from the zeta(ζ) chain of the antigen receptor and to a lesser extent CD3γ, δ, ε.

[0124] The cytoplasmic domain, as it exists naturally or as it may betruncated, modified or mutated will be at least about 10, usually atleast about 30 amino acids, more usually at least about 50 amino acids,and generally not more than about 400 amino acids, usually not more thanabout 200 amino adds. (See Romeo, et al., Cell (1992) 68, 889-893.)While any species can be employed, the species endogenous to the hostcell is usually preferred. However, in many cases, the cytoplasmicdomain from a different species can be used effectively. Any of theabove indicated cytoplasmic domains may be used, as well as others whichare presently known or may subsequently be discovered. TABLE 1 DNABinding Ligand Element Factor(s) Gene Reference Insulin cAMP LRFI jun-BMol. Cell Biol. and others responsive many (1992), 12, element genes4654, PNAS, (cre) 83, 3439 PDGF, SRE SRF/SR c-fos Mol. Cell Biol. FGF,TGF EBP (1992), 12, and others 4769 EGF VL30 RVL-3 Mol. Cell. Biol. RSRFvirus (1992), 12, c-jun 2793 do. (1992), 12, 4472 IFN-α ISRE ISGF-3 GeneDev. (1989) 3,1362 IFN-γ GAS GAF GBP Mol. Cell. Biol. (1991) 11, 182 PMAand AP-1 many Cell (1987) TCR genes 49, 729-739 TNF NFκB many Cell(1990) 62, 1019-1029 Antigen ARRE-1 OAP/O many Mol. Cell Biol. ct-1genes (1988) 8, 1715 Antigen ARRE-2 NFAT IL-2 Science (1988) enhancer241, 202

[0125] For the most part, the other chimeric proteins associated withtranscription factors, will differ primarily in having a cellulartargeting sequence which directs the chimeric protein to the internalside of the nuclear membrane and having transcription factors orportions thereof as the action domains. Usually, the transcriptionfactor action domains can be divided into “DNA binding domains” and“activation domains.” One can provide for a DNA binding domain with oneor more ligand binding domains and an activation domain with one or moreligand binding domains. In this way the DNA binding domain can becoupled to a plurality of binding domains and/or activation domains.Otherwise, the discussion for the chimeric proteins associated with thesurface membrane for signal transduction is applicable to the chimericproteins for direct binding to genomic DNA. Similarly, the chimericprotein associated with exocytosis will differ primarily as to theproteins associated with fusion of the vesicle membrane with the surfacemembrane, in place of the transducing cytoplasmic proteins.

[0126] B. Cellular Targeting Domains

[0127] A signal peptide or sequence provides for transport of a chimericprotein to the cell surface membrane, where the same or other sequencescan result in binding of the chimeric protein to the cell surfacemembrane. While there is a general motif of signal sequences, two orthree N-terminal polar amino acids followed by about 15-20 primarilyhydrophobic amino adds, the individual amino acids can be widely varied.Therefore, substantially any signal peptide can be employed which isfunctional in the host and may or may not be associated with one of theother domains of the chimeric protein. Normally, the signal peptide isprocessed and will not be retained in the mature chimeric protein. Thesequence encoding the signal peptide is at the 5′-end of the codingsequence and will include the initiation methionine codon.

[0128] The choice of membrane retention domain is not critical to thisinvention, since it is found that such membrane retention domains aresubstantially fungible and there is no critical amino acid required forbinding or bonding to another membrane region for activation. Thus, themembrane retention domain can be isolated from any convenient surfacemembrane or cytoplasmic protein, whether endogenous to the host cell ornot.

[0129] There are at least two different membrane retention domains: atransmerbrane retention domain, which is an amino acid sequence whichextends across the membrane, and a lipid membrane retention domainswhich lipid associates with the lipids of the cell surface membrane.

[0130] For the most part, for ease of construction the transmembranedomain of the cytoplasmic domain or the receptor domain can be employed,which may tend to simplify the construction of the fused protein.However, for the lipid membrane retention domain the processing signalwill usually be added at the 5′ end of the coding sequence forN-terminal binding to the membrane and, proximal to the 3′ end forC-terminal binding. The lipid membrane retention domain will have alipid of from about 12 to 24 carbon atoms, particularly 14 carbon atoms,more particularly myristoyl, joined to glycine. The signal sequence forthe lipid binding domain is an N-terminal sequence and can be variedwidely, usually having glycine at residue 2 and lysine or arginine atresidue 7 (Kaplan, et al., Mol. Cell. Biol. (1988) 8, 2435). Peptidesequences involving post-translational processing to provide for lipidmembrane binding are described by Carr, et al., PNAS USA (1988) 79,6128; Aitken, et al., FEBS Lett. (1982) 150, 314; Henderson, et al.,PNAS USA (1983) 80, 319; Schulz, et al., Virology (1984), 123, 2131;Dellman, et al., Nature (1985) 314, 374; and reviewed in Ann. Rev. ofBiochem. (1988) 57, 69. An amino acid sequence of interest includes thesequence M-G-S-S-K-S-K-P-K-D-P-S-Q-R. Various DNA sequences can be usedto encode such sequence in the fused receptor protein.

[0131] Generally, the transmembrane domain will have from about 18-30amino acids, more usually about 20-30 amino acids, where the centralportion will be primarily neutral, non-polar amino acids, and thetermini of the domain will be polar amino acids, frequently chargedamino acids, generally having about 1-2 charged, primarily basic aminoacids at the termnini of the transmembrane domain followed by a helicalbreak residue, e.g. pro- or gly-.

[0132] C. Ligand Binding Domain

[0133] The ligand binding (“dimerization” or “receptor”) domain of anyof the chimeric proteins of this invention can be any convenient domainwhich will allow for induction using, or bind to, a natural or unnaturalligand, preferably an unnatural synthetic ligand. The binding domain canbe internal or external to the cellular membrane, depending upon thenature of the construct and the choice of ligand. A wide variety ofbinding proteins, including receptors, are known, including bindingproteins associated with the cytoplasmic regions indicated above. Ofparticular interest are binding proteins for which ligands (preferablysmall organic ligands) are known or may be readily produced. Thesereceptors or ligand binding domains include the FKBPs and cyclophilinreceptors, the steriod receptors, the tetracycline receptor the otherreceptors indicated above, and the like, as well as “unnatural”receptors, which can be obtained from antibodies, particularly the heavyor light chain subunit, mutated sequences thereof, random amino acidsequences obtained by stochastic procedures, combinatorial syntheses,and the like. For the most part, the receptor domains will be at leastabout 50 amino acids, and fewer than about 350 amino acids, usuallyfewer than 200 amino acids, either as the natural domain or truncatedactive portion thereof. Preferably the binding domain will be small (<25kDa, to allow efficient transfection in viral vectors), monomeric,nonimmunogenic, and should have synthetically accessible, cellpermeable, nontoxic ligands that can be configured for dimerization.

[0134] The receptor domain can be intracellular or extracellulardepending upon the design of the construct encoding the chimeric proteinand the availability of an appropriate ligand. For hydrophobic ligands,the binding domain can be on either side of the membrane but forhydrophilic ligands, particularly protein ligands, the binding domainwill usually be external to the bell membrane, unless there is atransport system for internalizing the ligand in a form in which it isavailable for binding. For an intracellular receptor, the construct canencode a signal peptide and transmerbrane domain 5′ or 3′ of thereceptor domain sequence or by having a lipid attachment signal sequence5′ or 3′ of the receptor domain sequence. Where the receptor domain isbetween the signal peptide and the transmembrane domain, the receptordomain will be extracellular.

[0135] The portion of the construct encoding the receptor can besubjected to mutagenesis for a variety of reasons. The mutagenizedprotein can provide for higher binding affinity, allow fordiscrimination by the ligand of the naturally occurring receptor and themutagenized receptor, provide opportunities to design a receptor-ligandpair, or the like. The change in the receptor can involve changes inamino acids known to be at the binding site, random mutagenesis usingcombinatorial techniques, where the codons for the amino acidsassociated with the binding site or other amino acids associated withconformational changes can be subject to mutagenesis by changing thecodon(s) for the particular amino acid either with known changes orrandomly, expressing the resulting proteins in an appropriateprokaryotic host and then screening the resulting proteins for binding.Illustrative of this situation is to modify FKBP12's Phe36 to Ala and/orAsp37 to Gly or Ala to accommodate a substituent at positions 9 or 10 ofFK506 or FK520. In particular, mutant FKBP12 moieties which contain Val,Ala, Gly, Met or other small amino acids in place of one or more ofTyr26, Phe36, Asp37, Tyr82 and Phe99 are of particular interest asreceptor domains for FK506-type and FK520-type ligands containingmodifications at C9 and/or C10.

[0136] Antibody subunits, e.g. heavy or light chain, particularlyfragments, more particularly all or part of the variable region, orfusions of heavy and light chain to create high-affinity binding, can beused as the binding domain. Antibodies can be prepared against haptenicmolecules which are physiologically acceptable and the individualantibody subunits screened for binding affinity. The cDNA encoding thesubunits can be isolated and modified by deletion of the constantregion, portions of the variable region, mutagenesis of the variableregion, or the like, to obtain a binding protein domain that has theappropriate affinity for the ligand this way, almost any physiologicallyacceptable haptenic compound can be employed as the ligand or to providean epitope for the ligand. Instead of antibody units, natural receptorscan be employed, where the binding domain is known and there is a usefulligand for binding.

[0137] The ability to employ in vitro mutagenesis or combinatorialmodifications of sequences encoding proteins allows for the productionof libraries of proteins which can be screened for binding affinity fordifferent ligands. For example, one can totally randomize a sequence of1 to 5, 10 or more codons, at one or more sites in a DNA sequenceencoding a binding protein, make an expression construct and introducethe expression construct into a unicellular microorganism, and develop alibrary. One can then screen the library for binding affinity to one ordesirably a plurality of ligands. The best affinity sequences which arecompatible with the cells into which they would be introduced can thenbe used as the binding domain. The ligand would be screened with thehost cells to be used to determine the level of binding of the ligand toendogenous proteins. A binding profile could be defined weighting theratio of binding affinity to the mutagenized binding domain with thebinding affinity to endogenous proteins. Those ligands which have thebest binding profile could then be used as the ligand. Phage displaytechniques, as a non-limiting example, can be used in carrying out theforegoing.

[0138] D. Multimerization

[0139] The transduced signal will normally result from ligand-mediatedoligomerization of the chimeric protein molecules, i.e. as a result ofoligomerization following ligand binding, although other binding events,for example allosteric activation, can be employed to initiate thesignal. The construct of the chimeric protein will vary as to the orderof the various domains and the number of repeats of an individualdomain. For the extracellular receptor domain in the 5′-3′ direction oftransition, the construct will encode a protein comprising the signalpeptide, the receptor domain, the transmembrane domain and the signalinitiation domain, which last domain will be intracellular(cytoplasmic). However, where the receptor domain is intracellular,different orders may be employed, where the signal peptide can befollowed by either the receptor or signal initiation domain, followed bythe remaining domain, or with a plurality of receptor domains, thesignal initiation domain can be sandwiched between receptor domains.Usually, the active site of the signal initiation domain will beinternal to the sequence and not require a free carboxyl terminus.Either of the domains can be multimerized, particularly the receptordomain, usually having not more than about 5 repeats, more usually notmore than about 3 repeats.

[0140] For multimerizing the receptor, the ligand for the receptordomains of the chimeric surface membrane proteins will usually bemultimeric in the sense that it will have at east two binding sites,with each of the binding sites capable of binding to the receptordomain. Desirably, the subject ligands will be a dimer or higher orderoligomer, usually not greater than about tetrameric, of small syntheticorganic molecules, the individual molecules typically being at leastabout 150 D and fewer than about 5 kD, usually fewer than about 3 kD. Avariety of pairs of synthetic ligands and receptors can be employed. Forexample, in embodiments involving natural receptors, chimeric FK506 canbe used with an FKBP receptor, dimerized cyclosporin A can be used withthe cyclophilin receptor, dimerized estrogen with an estrogen receptor,dimerized glucocorticoids with a glucocorticoid receptor, dimerizedtetracycline with the tetracycline receptor, dimerized vitamin D withthe vitamin D receptor, and the like. Alternatively higher orders of theligands, e.g. trimeric can be used. For embodiments involving unnaturalreceptors, e.g. antibody subunits, modified antibody subunits ormodified receptors and the like, any of a large variety of compounds canbe used. A significant characteristic of these ligand units is that theybind the receptor with high affinity (preferably with a K_(d)≦10⁻⁸ M)and are able to be dimerized chemically.

[0141] The ligand can have different receptor binding molecules withdifferent epitopes (also referred to as “HED” reagents, since they canmediate hetero-dimerization or hetero-oligomerization of chimericproteins having the same or different binding domains. For example, theligand may comprise FK506 or an FK506-type moiety and a CsA or acyclosporin type moiety. Both moieties are covalently attached to acommon linker moiety. Such a ligand would be useful for mediating theoligomerization of a first and second chimeric protein where the firstchimeric protein contains a receptor domain such as an FKBP12 which iscapable of binding to the FK506-type moiety and the second chimericprotein contains a receptor domain such as cyclophilin which is capableof binding to the cyclosporin A-type moiety.

[0142] C. Tissue Specific Expression of the Chimeric Proteins

[0143] It will be preferred in certain embodiments, that apoptosisand/or optional biological events (e.g. the expression of a target gene)be triggered in a cell-specific or tissue-specific manner. To achievesuch specificity, one may render the expression of the chimeric proteinscell-type specific. Such specificity of expression may be achieved bylinking one or more of the DNA sequences encoding the chimeric proteins)to a cell-type specific transcriptional regulatory sequence (e.g.promoter/enhancer). Numerous cell-type specific transcriptionalregulatory sequences are known. Others may be obtained from genes whichare expressed in a cell-specific manner.

[0144] For example, constructs for expressing the chimeric proteins maycontain regulatory sequences derived from genes known for specificexpression in selected tissues. Representative illustrative examples aretabulated below: Tissue Gene Reference lens γ2-crystallin Breitman, M.L., Clapoff, S., Rossant, J., Tsui, L. C., Golde, L. M., Maxwell, L. H.,Bernstin, A. (1987) Genetic Ablation: targeted expression of a toxingene causes microphthalmia in transgenic mice. Science 238: 1563-1565αA-crystallin Landel, C. P., Zhao, J., Bok, D., Evans, G. A. (1988)Lens-specific expression of a recombinant ricin induces developmentaldefects in the eyes of transgenic mice. Genes Dev. 2: 1168-1178 Kaur,S., key, B., Stock, J., McNeish, J. D., Akeson, R, Potter, S. S. (1989)Targeted ablation of alpha-ystallin- synthesizing cells produces lens-deficient eyes in transgenic mice. Development 105: 613-619 pituitaxy-Growth hormone Behringer, R. R., Mathews, L. S., somatrophic Palmiter,R. D., Brinster, R. L. (1988) cells Dwarf mice produced by geneticablation of growth hormone-expressing cells. Genes Dev. 2: 453-461pancreas Insulin- Ornitz, D. M., Palmiter, R. D., Elastase-acinarHammer, R. E., Brinster, R.L, Swift, cell Specific G. H., MacDonald, R.J. (1985) Specific expression of an elastase- human growth fusion inpancreatic acinar cells of transgeneic mice. Nature 131: 600-603Palmiter, R. D., Behringer, R. R., Quaife, C. J., Maxwell, F., Maxwell,I. H., Brinster, R. L. (1987) Cell lineage ablation in transgeneic miceby cell-specific expression of a toxin gene. Cell 50: 435-443 T cellslck promoter Chaffin, K. E., Beals, C. R., Wilkie, T .M., Forbush, K.A., Simon, M.I., Perlmutter, R.M. (1990) EMBO Journal 9: 3821-3829 Bcells Immunoglobulin Borelli, E., Heyman, R., Hsi, M., kappa lightEvans, R. M. (1988) Targeting of an chain inducible toxic phenotype inanimal cells. Proc. Natl. Acad. Sci. USA 85: 7572-7576 Heyman, R. A.,Borrelli, E., Lesley, J., Anderson, D., Richmond,, D. D., Baird, S. M.,Hyman, R., Evans, R.M. (1989) Thymidine kinase obliteration: creation oftransgenic mice with controlled immunodeficiencies. Proc. Natl. Acad.Sci. USA 86: 2698-2702 Schwann P_(O) promoter Messing. A., Behringer, R.R., cells Hammang, J. P. Palmiter, R. D., Brinster, R. L., Lemke, G.,P_(O) promoter directs espression of reporter and toxin genes toSchwann cells of transgenic mice. Neuron 8: 507-520 1992 Myelin basicMiskimins, R. Knapp, L., Dewey, M. protein J., Zhang, X. Cell andtissue-specific expression of a heterologous gene under control of themyelin basic protein gene promoter in trangenic mice. Brain Res DevBrain Res 1992 Vol 65: 217-21 spermatids protamine Breitman, M. L.,Rombola, H., Maxwell, I. H., Klintworth, G.K., Bernstein, A. (1990)Genetic ablation in transgenic mice with attenuated diphtheria toxin Agene. Mol. Cell. Biol. 10: 414-479 lung Lung surfacant Ornitz, D. M.,Palmiter, R. D., gene Hammer, R. E., Brinster, R. L., Swift, G. H.,MacDonald, R. J. (1985) Specific expression of an elastase- human growthfusion in pancreatic acinar cells of transgeneic mice. Nature 131:600-603 adipocyte P2 Ross, S. R., Braves, R. A., Spiegelman, BM Targetedexpression of a toxin gene to adipose tissue: transgenic mice resistantto obesity Genes and Dev 7: 1318-24 1993 muscle myosin light Lee, K.J.,Ross, R. S., Rockman, chain H. A., Harris, A. N., O'Brien, T. X.,van-Bilsen, M., Shubeita, H. E., Kandolf, R., Brem, G., Prices et alJ.BIol. Chem. 1992 Aug 5, 267: 15875-85 Alpha actin Muscat, GE., Peny, S.,Prentice, H. Kedes, L. The human skeletal alpha- actin gene is regulatedby a muscle- specific enhancer that binds three nuclear factors. GeneExpression 2, 111-26, 1992 neurons neurofilament Reeben, M. Halmekyto,M. Alhonen, L. proteins Sinervirta, R. Saarma, M. Janne, J.Tissue-specific expression of rat light neurofilament promoter- drivenreporter gene in transgenic mice. BBRC 1993: 192: 465-70 liver tyrosineaminotransferase, albumin, apolipoproteins

[0145] Identification of Tissue Specific Promoters

[0146] To identify the sequences that control the tissue- or cell-typespecific expression of a gene, one isolates a genomic copy of theselected gene including sequences “upstream” from the exons that codefor the protein.      5′flanking sequences       coding sequencesI-------------------------------------I=======================I

[0147] These upstream sequences are then usually fused to an easilydetectable reporter gene like beta-galactosidase, in order to be able tofollow the expression of the gene under the control of upstreamregulatory sequences.           5′flanking sequences       reporter geneI-------------------------------------I=-=-=-=-=-=-=-=-=-=-I

[0148] To establish which upstream sequences are necessary andsufficient to control gene expression in a cell-type specific manner,the complete upstream sequences are introduced into the cells ofinterest to determine whether the initial done contains the controlsequences. Reporter gene expressor is monitored as evidence ofexpression. I----------------------------------I=-=-=-=-=-=-=-=-=-=-II-------------------------I=-=-=-=-=-=-=-=-=-=-II---------------I=-=-=-=-=-=-=-=-=-=-I I-------I=-=-=-=-=-=-=-=-=-=-II-    ---------------------I=-=-=-=-=-=-=-=-=-=-II-      -------------------I=-=-=-=-=-=-=-=-=-=-II-                   ------------I=-=-=-=-=-=-=-=-=-=-II-                           ----I=-=-=-=-=-=-=-=-=-=-

[0149] If these sequences contain the necessary sequences for cell-typespecific expression, deletions may be made in the 5′ flanking sequencesto determine which sequences are minimally required for cell-typespecific expression. This can be done by making transgenic mice witheach construct and monitoring beta gal expression, or by first examiningthe expression in specific culture cells, with comparison to expressionin non-specific cultured cells.

[0150] Several successive rounds of deletion analysis normally pinpointthe minimal sequences required for tissue specific expression.Ultimately, these sequences are then introduced into transgenic mice toconfirm that the expression is only detectable in the cells of interest.

[0151] VI. Cells

[0152] The cells may be procaryotic, but are preferably eucaryotic,including plant, yeast, worm, insect and mammalian. At present it isespecially preferred that the cells be mammalian, particularly primate,more particularly human, but can be associated with any animal ofinterest, particularly domesticated animals, such as equine, bovine,murine, ovine, canine, feline, etc. Among these species, various typesof cells can be involved such as hematopoietic, neural, mesenchymal,cutaneous, mucosal, stromal, muscle, spleen, reticuloendothelial,epithelial, endothelial, hepatic, kidney, gastrointestinal, pulmonary,etc. Of particular interest are hematopoietic cells, which include anyof the nucleated cells which may be involved with the lymphoid ormyelomonocytic lineages. Of particular interest are members of the T-and B-cell lineages, macrophages and monocytes, myoblasts andfibroblasts. Also of particular interest are stem and progenitor cells,such as hematopoietic neural, stromal, muscle, hepatic, pulmonary,gastrointestinal, etc.

[0153] The cells can be autologous cells, syngeneic cells, allogeniccells and even in some cases, xenogeneic cells. The cells may bemodified by changing the major histocompatibility complex (“MHC”)profile, by inactivating β₂-microglobulin to prevent the formation offunctional Cass I MHC molecules, inactivation of Class II molecules,providing for expression of one or more MHC molecules, enhancing orinactivating cytotoxic capabilities by enhancing or inhibiting theexpression of genes associated with the cytotoxic activity, or the like.

[0154] In some instances specific clones or oligoclonal cells may be ofinterest, where the cells have a particular specificity, such as T cellsand B cells having a specific antigen specificity or homing target sitespecificity.

[0155] VII. Ligands

[0156] A wide variety of ligands, including both naturally occurring andsynthetic substances, can be used in this invention to effectoligomerization of the chimeric protein molecules. Applicable andreadily observable or measurable criteria for selecting a ligand are:(A) the ligand is physiologically acceptable (i.e., lacks undue toxicitytowards the cell or animal for which it is to be used), (B) it has areasonable therapeutic dosage range, (C) desirably (for applications inwhole animals, including gene therapy applications), it can be takenorally (is stable in the gastrointestinal system and absorbed into thevascular system), (D) it can cross the cellular and other membranes, asnecessary, and (E) binds to the receptor domain with reasonable affinityfor the desired application. A first desirable criterion is that thecompound is relatively physiologically inert, but for its activatingcapability with the receptors. The less the ligand binds to nativereceptors and the lower the proportion of total ligand which binds tonative receptors, the better the response will normally be.Particularly, the ligand should not have a strong biological effect onnative proteins. For the most part, the ligands will be non-peptide andnon-nucleic acid.

[0157] The subject compounds will for the most part have two or moreunits, where the units can be the same or different, joined togetherthrough a central linking group. The “units” will be individual moieties(e.g., FK506, FK520, cyclosporin A, a steroid, etc.) capable of bindingthe receptor domain. Each of the units will usually be joined to thelinking group through the same reactive moieties, at least in homodimersor higher order homo-oligomers.

[0158] As indicated above, there are a variety of naturally-occurringreceptors for small non-proteinaceous organic molecules, which smallorganic molecules fulfill the above criteria, and can be dimerized atvarious sites to provide a ligand according to the subject invention.Substantial modifications of these compounds are permitted so long asthe binding capability is retained and with the desired specificity.Many of the compounds will be macrocyclics, e.g. macrolides. Suitablebinding affinities will be reflected in Kd values well below 10⁻⁴,preferably below 10⁻⁶, more preferably below about 10⁻⁷, althoughbinding affinities below 10⁻⁹ or 10⁻¹⁰ are possible, and in some caseswill be most desirable.

[0159] Currently preferred ligands comprise oligomers, usually dimers,of compounds capable of binding to an FKBP protein and/or to acyclophilin protein. Such ligands includes homo- and heteromultimers(usually 2-4, more usually 2-3 units) of cyclosporin A, FK506, FK520,and rapamycin, and derivatives thereof, which retain their bindingcapability to the natural or mutagenized binding domain. Manyderivatives of such compounds are already known, including synthetichigh affinity FKBP ligands, which can be used in the practice of thisinvention. See e.g. Holt et al, J Am Chem Soc 1993, 115, 9925-9935.Sites of interest for linking of FK506 and analogs thereof includepositions involving annular carbon atoms from about 17 to 24 andsubstituent positions bound to those annular atoms, e.g. 21 (allyl), 22,37, 38, 39 and 40, or 32 (cyclohexyl), while the same positions exceptfor 21 are of interest for FK520. For cyclosporin, sites of interestinclude MeBmt, position 3 and position 8.

[0160] Of particular interest are modifications to the ligand whichchange its binding characteristics, particularly with respect to theligand's naturally occurring receptor. Concomitantly, one would changethe binding protein to accommodate the change in the ligand. For exampleone can modify the groups at position 9 or 10 of FK506(see Van Duyne etal (1991) Science 252, 839), so as to increase their steric requirement,by replacing the hydroxyl with a group having greater stericrequirements, or by modifying the carbonyl at position 10, replacing thecarbonyl with a group having greater steric requirements orfunctonalizing the carbonyl, e.g. forming an N-substituted Schiff's baseor imine, to enhance the bulk at that position. Various functionalitieswhich can be conveniently introduced at those sites are all groups toform ethers, acylamido groups, N-alkylated amines, where a2-hydroxyethylimine can also form a 1,3-oxazoline, or the like.Generally, the substituents will be from about 1 to 6, usually 1 to 4,and more usually 1 to 3 carbon atoms, with from 1 to 3, usually 1 to 2heteroatoms, which will usually be oxygen, sulfur, nitrogen, or thelike. By using different derivatives of the basic structure, one cancreate different ligands with different conformational requirements forbinding. By mutagenizing receptors, one can have different receptors ofsubstantially the same sequence having different affinities for modifiedligands not differing significantly in structure.

[0161] Other ligands which can be used are steroids. The steroids can beoligomerized, so that their natural biological activity is substantiallydiminished without loss of their binding capability with respect to achimeric protein containing one or more steroid receptor domains. By wayof non-limiting example, glucocorticoids and estrogens can be so used.Various drugs can also be used, where the drug is known to bind to aparticular receptor with high affinity. This is particularly so wherethe binding domain of the receptor is known, thus permitting the use inchimeric proteins of this invention of only the binding domain, ratherthan the entire native receptor protein. For this purpose, enzymes andenzyme inhibitors can be used.

[0162] A. Linkers

[0163] Various functionalities can be involved in the linking, such asamide groups, including carbonic add derivatives, ethers, esters,including organic and inorganic esters, amino, or the like. To providefor linking, the particular monomer can be modified by oxidation,hydroxylation, substitution, reduction, etc., to provide a site forcoupling. Depending on the monomer, various sites can be selected as thesite of coupling.

[0164] The multimeric ligands can be synthesized by any convenientmeans, where the linking group will be at a site which does notinterfere with the binding of the binding site of a ligand to thereceptor. Where the active site for physiological activity and bindingsite of a ligand to the receptor domain are different, it will usuallybe desirable to link at the active site to inactivate the ligand.Various linking groups can be employed, usually of from 1-30, moreusually from about 1-20 atoms in the chain between the two molecules(other than hydrogen), where the linking groups will be primarilycomposed of carbon, hydrogen, nitrogen, oxygen, sulphur and phosphorous.The linking groups can involve a wide variety of functionalities, suchas amides and esters, both organic and inorganic, amines, ethers,thioters, disulfides, quaternary ammonium salts, hydrazines, etc. Thechain can include alphatic, alicyclic, aromatic or heterocyclic groups.The chain will be selected based on ease of synthesis and the stabilityof the multimeric ligand. Thus, if one wishes to maintain long-termactivity, a relatively inert chain will be used, so that the multimericligand link will not be cleaved. Alternatively, if one wishes only ashort half-life in the blood stream, then various groups can be employedwhich are readily cleaved, such as esters and amides, particularlypeptides, where circulating and/or intracellular proteases can cleavethe linking group.

[0165] Various groups can be employed as the lining group betweenligands, such as alkylene, usually of from 2 to 20 carbon atoms,azalkylene (where the nitrogen will usually be between two carbonatoms), usually of from 4 to 18 carbon atoms), N-alkylene azalkylene(see above), usually of from 6 to 24 carbon atoms, arylene, usually offrom 6 to 18 carbon atoms, ardialkylene, usually of from 8 to 24 carbonatoms, bis-carboxamido alkylene of from about 8 to 36 carbon atoms, etc.Illustrative groups include decylene, octadecylene, 3-azapentylene,5-azadecylene, N-butylene 5-azanonylene, phenylene, xylylene,p-dipropylenebenzene, bis-benzoyl 1,8-diaminooctane and the like.Multivalent or other (see below) ligand molecules containing linkermoieties as described above can be evaluated with chimeric proteins ofthis invention bearing corresponding receptor domains using materialsand methods described in the examples which follow.

[0166] B. Ligand Characteristics

[0167] For intracellular binding domains, the ligand will be selected tobe able to be transferred across the membrane in a bioactive forms thatis, it will be membrane permeable. Various ligands are hydrophobic orcan be made so by appropriate modification with lipophilic groups.Particularly, the linking bridge can serve to enhance the lipophilicityof the ligand by providing aliphatic side chains of from about 12 to 24carbon atoms. Alternatively, one or more groups can be provided whichwill enhance transport across the membrane, desirably without endosomeformation.

[0168] In some instances, multimeric ligands need not be employed. Forexample, molecules can be employed where two different binding sitesprovide for dimerization of the receptor. In other instances, binding ofthe ligand can result in a conformational change of the receptor domain,resulting in activation, e.g. oligomerization, of the receptor. Othermechanisms may also be operative for inducing the signal, such asbinding a single receptor with a change in conformation resulting inactivation of the cytoplasmic domain.

[0169] As discussed elsewhere, ligands capable of initiating any of theoptional additional cellular processes, such as transcription of atarget gene, should preferably be selected so as not to cross react withthe primary chimeric proteins in the engineered cells to initiateapoptosis.

[0170] C. Ligand Antagonists

[0171] Monomeric ligands can be used for reversing the effect of themultimeric ligand, i.e., for preventing, inhibiting or disruptingoligomer formation or maintenance. Thus, if one wishes to rapidlyterminate the effect of cellular activation, a monomeric ligand can beused. Conveniently, the parent ligand moiety on be modified at the samesite as the multimer using the same procedure, except substituting amonofunctional compound for the polyfunctional compound. Instead of thepolyamines, monoamines, particularly of from 2 to 20 (although they canbe longer), and usually 2 to 12, carbon atoms can be used, such asethylamine, hexylamine, benzylamine, etc. Alternatively, the monovalentparent compound can be used, in cases (or at dosage levels) in which theparent compound does not have undue undesirable physiological activity(e.g. immunosuppression, mitogenesis, toxicity, etc.)

[0172] D. Illustrative Hetero-Oligomerizing (HED) and Homo-Oligomerizing(HOD) Reagents with “Bumps” that can Bind to Mutant Receptors ContainingCompensatory Mutations

[0173] As discussed above, one can prepare modified HED/HOD reagentsthat will fail to bind appreciably to their wild type receptors (e.g.,FKBP12) due to the presence of substituents (“bumps”) on the reagentsthat sterically clash with sidechain residues in the receptor's bindingpocket. One may also make corresponding receptors that contain mutationsat the interfering residues (“compensatory mutations”) and thereforegain the ability to bind ligands with bumps. Using “bumped” ligandmoieties and receptor domains bearing “compensatory mutations” shouldenhance the specificity and thus the potency of our reagents. Bumpedreagents should not bind to the endogenous, wild type receptors, whichcan otherwise act as a “buffer” toward dimerizers based on naturalligand moieties. In addition, the generation of novel receptor-ligandpairs should simultaneously yield the HED reagents that will be usedwhen heterodimerization is required. For example, regulated vesiclefusion may be achieved by inducing the heterodimerization of syntaxin (aplasma membrane fusion protein) and synaptobrevin (a vesicle membranefusion protein) using a HED reagent. This would not only provide aresearch tool, but could also serve as the basis of a gene therapytreatment for diabetes, using appropriately modified secretory cells.

[0174] As an illustration of “Bumped FK1012s” we prepared C10 acetamideand formamide derivatives of FK506. See FIG. 16 and our report, Spenceret al, “Controlling Signal Transduction with Synthetic Ligands,” Science262(1993): 1019-1024 for additional details concerning the syntheses ofFK1012s A-C and FK506M. We chose to create two classes of bumped K1012s:one with a bump at C10 and one at C9. The R- and S-isomers of the C10acetamide and formamide of FK506 have been synthesized according to thereaction sequence in FIG. 16B. These bumped derivatives have lost atleast three orders of magnitude in their binding affinity towards FKBP12(FIG. 16B). The affinities were determined by measuring the ability ofthe derivatives to inhibit FKBP12's rotamase activity.

[0175] An illustrative member of a second class of C9bumped derivativesis the spiro-epoxide (depicted in FIG. 16C), which has been prepared byadaptation of known procedures. See e.g. Fisher et al, J Org Chem 568(1991): 2900-7 and Edmunds et al, Tet Lett 32 48 (1991):819-820. Aparticularly interesting series of C9 derivatives are characterized bytheir sp³ hybridization and reduced oxidation state at C9. Several suchcompounds have been synthesized according to the reactions shown in FIG.16C.

[0176] It should be appreciated that heterodimers (and otherhetero-oligomerizers) must be constructed differently than thehomodimers, at least for applications where homodimer contaminationcould adversely affect their successful use. One illustrative syntheticstrategy developed to overcome this problem is outlined in FIG. 16D.Coupling of mono alloc-protected 1,6-hexanediamine (Stahl et al, J OrgChem 43 11 (1978): 2285-6) with a derivatized form of FK506 in methylenechloride with an excess of triethylamine gave an alloc-amine-substitutedFK506 in 44% yield. This intermediate can now be used in the couplingwith any activated FK506 (or bumped-FK506) molecule. Deprotection withcatalytic tetrakis-triphenylphosphine palladium in the presence ofdimedone at room temperature in THF removes the amine protecting group.Immediate treatment with an activated FK506 derivative, followed bydesilylation leads to a chimeric product. This technique has been usedto synthesize the illustrated HOD and HED reagents.

[0177] E. Illustrative Cyclosporin-Based Reagents

[0178] Cyclosporin A (CsA) is a cyclic undecapeptide that binds withhigh affinity (6 nM) to its intracellular receptor cyclophilin, an 18kDa monomeric protein. The resulting complex, like the FKBP12-FK506complex, binds to and inactivates the protein phosphatase calcineurinresulting in the immunosuppressive properties of the drug. As a furtherillustration of this invention, we have dimerized CsA via its MeBmt1sidechain in 6 steps and 35% overall yield to give (CsA)2 (FIG. 17,steps 1-4 were conducted as reported in Eberle et al, J Org Chem 57 9(1992): 2689-91). As with FK1012s, the site for dimerization was chosensuch that the resulting dimer can bind to two molecules of cyclophilinyet cannot bind to calcineurin following cyclophilin binding. We havedemonstrated that (CsA)2 binds to cyclophilin A with 1:2 stoichiometry.Hence, (CsA)2, like FK1012s, does not inhibit signaling pathways and isthus neither immunosuppressive nor toxic at concentrations useful forpracticing this invention.

[0179] VIII. Target Gene

[0180] A. Transcription Initiation Region

[0181] Target gene constructs will have a responsive element in the 5′region, which responds to ligand-mediated oligomerization of thechimeric receptor protein, presumably via the generation andtransduction of a transcription initiation signal as discussed ifra.Therefore, it will be necessary to select at least one transcriptioninitiation system, e.g. transcription factor, which is activated eitherdirectly or indirectly, by the cytoplasmic domain or can be activated byassociation of two domains. It will also be necessary to select at leastone promoter region which is responsive to the resulting transcriptioninitiation system. Either the promoter region or the gene under itstranscriptional control need be selected. In other words, an actiondomain can be selected for the chimeric proteins (encoded by a firstsseries construct) based on the role of that action domain in initiatingtranscription via a given promoter or responsive element. See e.g.Section V(A) “Cytoplasmic domains”, above.

[0182] Where the responsive element is known, it can be included in thetarget gene construct to provide an expression cassette for integrationinto the genome (whether episomally or by chromosomal incorporation). Itis not necessary to have isolated the particular sequence of theresponsive element, so long as a gene is known which istranscriptionally activated by the cytoplasmic domain upon naturalligand binding to the protein comprising the cytoplasmic domain.Homologous recombination could then be used for insertion of the gene ofinterest downstream from the promoter region to be under thetranscriptional regulation of the endogenous promoter region. Where thespecific responsive element sequence is known, that can be used inconjunction with a different transcription initiation region, which canhave other aspects such as a high or low activity as to the rate oftranscription, binding of particular transcription factors and the like.

[0183] The expression construct will therefore have at its 5′ end in thedirection of transcription, the responsive element and the promotersequence which allows for induced transcription initiation of a targetgene of interest, usually a therapeutic gene. The transcriptionaltermination region is not as important, and can be used to enhance thelifetime of or make short half-lived mRNA by inserting AU sequenceswhich serve to reduce the stability of the mRNA and, therefore, limitthe period of action of the protein. Any region can be employed whichprovides for the necessary transcriptional termination, and asappropriate, translational termination.

[0184] The responsive element can be a single sequence or can beoligomerized, usually having not more than about 5 repeats, usuallyhaving about 3 repeats.

[0185] Homologous recombination can also be used to remove or inactivateendogenous transcriptional control sequences, including promoter and/orresponsive elements, which are responsive to the oligomerization event,and/or to insert such responsive transcriptional control sequencesupstream of a desired endogenous gene.

[0186] B. Product

[0187] A wide variety of genes can be employed as the target gene,including genes that encode a protein of interest or an antisensesequence of interest or a ribozyme of interest. The target gene can beany sequence of interest which provides a desired phenotype. The targetgene can express a surface membrane protein a secreted protein, acytoplasmic protein, or there can be a plurality of target genes whichcan express different types of products. The target gene may be anantisense sequence which can modulate a particular pathway by inhibitinga transcriptional regulation protein or turn on a particular pathway byinhibiting the translation of an inhibitor of the pathway. The targetgene can encode a ribozyme which may modulate a particular pathway byinterfering, at the RNA level, with the expression of a relevanttranscriptional regulator or with the expression of an inhibitor of aparticular pathway. The proteins which are expressed, singly or incombination can involve homing, cytotoxicity, proliferation, immuneresponse, inflammatory response, clotting or dissolving of clots,hormonal regulation, or the like. The proteins expressed could benaturally-occurring, mutants of naturally-occurring proteins, uniquesequences, or combinations thereof.

[0188] The gene can be any gene which is secreted by a so that theencoded product can be made available at will, whenever desired orneeded by the host. Various secreted products include hormones, such asinsulin, human growth hormone, glucagon, pituitary releasing factor,ACTH, melanotropin, relaxin, etc.; growth factors, such as EGF, IGF-1,TGF-α, -β, PDGF, G-CSF, M-CSF, GM-CSF, FGF, erythropoietin,megakaryocytic stimulating and growth factors, etc.; interleukins, suchas IL-1 to -13; TNF-α and -β, etc.; and enzymes, such as tissueplasminogen activator, members of the complement cascade, perforins,superoxide dismutase, coagulation factors, antithrombin-III, FactorVIIc, Factor VIIIvW, α-anti-trypsin, protein C, protein S, endorphins,dynorphin, bone morphogenetic protein, CFTR, etc.

[0189] The gene can be any gene which is naturally a surface membraneprotein or made so by introducing an appropriate signal peptide andtransmembrane sequence. Various proteins include homing receptors, e.g.L-selectin (Mel-14), blood-related proteins, particularly having akringle structure, e.g. Factor VIIIc, Factor VIIIvW, hematopoietic cellmarkers, e.g. CD3, CD4, CD8, B cell receptor, TCR subunits α, β, γ, δ,CD10, CD19, CD28, CD33, CD38, CD41, etc., receptors, such as theinterleukin receptors IL-2R, IL-4R, etc., channel proteins, for influxor efflux of ions, e.g. H⁺, Ca⁺², K⁺, Na⁺, Cl⁻, etc., and the like;CFTR, tyrosine activation motif, ζ activation protein, etc.

[0190] Proteins may be modified for transport to a vesicle forexocytosis. By adding the sequence from a protein which is directed tovesicles, where the sequence is modified proximal to one or the otherterminus, or situated in an analogous position to the protein source,the modified protein will be directed to the Golgi apparatus forpackaging in a vesicle. This process in conjunction with the presence ofthe chimeric proteins for exocytosis allows for rapid transfer of theproteins to the extracellular medium and a relatively high localizedconcentration.

[0191] Also, intracellular proteins can be of interest, such as proteinsin metabolic pathways, regulatory proteins, steroid receptors,transcription factors, etc., particularly depending upon the nature ofthe host cell. Some of the proteins indicated above can also serve asintracellular proteins.

[0192] The following are a few illustrations of different genes. InT-cells, one may wish to introduce genes encoding one or both chains ofa T-cell receptor. For B-cells, one could provide the heavy and lightchains for an immunoglobulin for secretion. For cutaneous cells, e.g.keratinocytes, particularly stem cells keratinocytes, one could providefor infectious protection by secreting α-, β- or -γ interferon,antichemotactic factors, proteases specific for bacterial cell wallproteins, etc.

[0193] In addition to providing for expression of a gene havingtherapeutic value, there will be many situations where one may wish todirect a cell to a particular site. The site can include anatomicalsites, such as lymph nodes, mucosal tissue, skin, synovium, lung orother internal organs or functional sites, such as clots, injured sites,sites of surgical manipulation, inflammation, infection, etc. Byproviding for expression of surface membrane proteins which will directthe host cell to the particular site by providing for binding at thehost target site to a naturally-occurring epitope, localizedconcentrations of a secreted product can be achieved. Proteins ofinterest include homing receptors, e.g. L-selectin, GMP140, CLAM-1,etc., or addressins, e.g. ELAM-1, PNAd, LNAd, etc., clot bindingproteins, or cell surface proteins that respond to localized gradientsof chemotactic factors. There are numerous situations where one wouldwish to direct cells to a particular site, where release of atherapeutic product could be of great value.

[0194] In many situations one may wish to be able to kill the modifiedcells, e.g. where one wishes to terminate treatment with the cells,where the cells have become undesirable e.g. neoplastic, or where thepurpose served by the cells has already been served and their continuedpresence is undesirable. Modified cells of this invention, which arecapable of expressing a primary chimeric protein containing a domainsuch as the cytoplasmic domain of the Fas antigen or TNF receptor(Watanable-Fukunaga et al. Nature (1992) 356, 314-317), are readilyeliminated through apoptosis following exposure of the cells to a ligandcapable of oligomerizing the primary chimeras. Constructs encoding theprimary chimera may be designed for constitutive expression usingconventional materials and methods, so that the modified cells have suchproteins on their surface or present in their cytoplasm. Alternatively,one can provide for controlled expression, where the same or differentoligomerizing ligand can initiate expression of the primary chimera andinitiate apoptosis. By providing for the cytoplasmic portions of the Fasantigen or TNF receptor in the cytoplasm joined to binding regionsdifferent from the binding regions associated with expression of atarget gene of interest, one can kill the modified cells undercontrolled conditions.

[0195] C. Illustrative Exemplifications

[0196] By way of illustration, cardiac patients or patients susceptibleto stroke may be treated as follows. Cells modified as described hereinmay be administered to the patient and retained for extended periods oftime. Illustrative cells include plasma cells, B-cells, T-cells, orother hematopoietic cells. The cell would be modified to express aprotein which binds to a blood clot, e.g. having a kringle domainstructure or an adhesive interactive protein, e.g. CD41, and to expressa clot dissolving protein e g. tissue plasminogen activator,streptokinase, etc. In this way, upon ligand-mediated oligomerization,the cells would accumulate at the site of the dot and provide for a highlocalized concentration of the thrombolytic protein.

[0197] Another example is reperfusion injury. Cells of limited lifetimecould be employed, e.g. macrophages or polymorphonuclear leukocytes(“neutrophils”). The cells would have a neutrophil homing receptor todirect the cells to a site of reperfusion injury. The cell would alsoexpress superoxide dismutase, to destroy singlet oxygen and inhibitradical attack on the tissue.

[0198] A third example is autoimmune disease. Cells of extendedlifetime, e.g. T cells could be employed. The constructs would providefor a homing receptor for homing to the site of autoimmune injury andfor cytotoxic attack on cells causing the injury. The therapy would thenbe directed against cells causing the injury. Alternatively, one couldprovide for secretion of soluble receptors or other peptide or protein,where the secretion product would inhibit activation of the injurycausing cells or induce anergy. Another alternative would be to secretean antiinflammatory product, which could serve to diminish thedegenerative effects.

[0199] A fourth example involves treatment of chronic pain withendorphin via encapsulation. A stock of human fibroblasts is transfectedwith a construct in which the chimeric transcriptional regulatoryprotein controls the transcription of human endorphin. The DNA constructconsists of three copies of the binding site for the HNF-1*transcription factor GTTAAGTTAAC upstream of a TATAAA site and atranscriptional initiation site. The endorphin cDNA would be inserteddownstream of the initiation site and upstream of a polyadenylation andtermination sequences. Optionally, the endorphin cDNA is outfitted with“PEST” sequences to make the protein unstable or AUUA sequences in the3′ nontranslated region of the mRNA to allow it to be degraded quickly.

[0200] The fibroblasts are also transfected with a construct having twotransition units, one of which would encode the HNF-1* cDNA truncated toencode lust the DNA binding sequences from amino acids 1 to 250 coupledto a trimeric FKBP binding domain under the transcriptional andtranslational control of regulatory initiation and termination regionsfunctional in the fibroblasts. The construct would include an additionaltranscription unit driven by the same regulatory regions directing theproduction of a transitional activation domain derived from HNF-4coupled to trimeric FKBP. (The prime intends an altered FKBP that bindsat nM concentration to a modified FK506. The modification inhibitsbinding to the endogenous FKBP.)

[0201] These genetically modified cells would be encapsulated to inhibitimmune recognition and placed under the patient's skin or otherconvenient internal site. When the patient requires pain medication, thepatient administers a dimeric ligand FK506-FK506′, where about 1 μg to 1mg would suffice. In this manner one could provide pain relief withoutinjections or the danger of addiction.

[0202] A fifth example is the treatment of osteoporosis. Lymphocytes canbe clonally developed or skin fibroblasts grown in culture from thepatient to be treated. The cells would be transfected as describedabove, where a bone morphogenic factor cDNA gene would replace theendorphin gene. For lymphocytes, antigen specific clones could be usedwhich would allow their destruction with antibodies to the idiotype ofthe sIg. In addition, administration of the antigen for the sIg wouldexpand the cell population to increase the amount of the protein whichcould be delivered. The lymphocyte clones would be infused and theligand administered as required for production of the bone morphogenicfactor. By monitoring the response to the ligand, one could adjust theamount of bone morphogenic factor which is produced, so as to adjust thedosage to the required level.

[0203] Another situation is to modify antigen specific T cells, whereone can activate expression of a protein product to activate the cells.The T cell receptor could be directed against tumor cells, pathogens,cells mediating autoimmunity, and the like. By providing for activationof the cells, for example, an interleukin such as IL-2, one couldprovide for expansion of the modified T cells in response to a ligand.Other uses of the modified T cells would include expression of homingreceptors for directing the T cells to specific sites, wherecytotoxicity, upregulation of a surface membrane protein of targetcells, e.g. endothelial cells, or other biological event would bedesired.

[0204] Alternatively one may want to deliver high doses of cytotoxicfactors to the target site. For example, upon recognition of tumorantigens via a homing receptor, tumor-infiltrating lymphocytes (TILs)may be triggered to deliver toxic concentrations of TNF or other similarproduct.

[0205] Another alternative is to export hormones or factors which areexocytosed. By providing for enhanced exocytosis, a greater amount ofthe hormone or factor will be exported; in addition, if there is afeedback mechanism based on the amount of the hormone or factor in thecytoplasm, increased production of the hormone or factor will result.Or, one may provide for induced expression of the hormone or factor, sothat expression and export may be induced concomitantly.

[0206] One may also provide for proteins in retained body fluids, e g.vascular system, lymph system, cerebrospinal fluid, etc. By modifyingcells which can have an extended lifetime in the host, e.g.hematopoietic cells, keratinocytes, muscle cells, etc. particularly,stem cells, the proteins can be maintained in the fluids for extendedperiods of time. The cells may be modified with constructs which providefor secretion or endocytosis. The constructs for secretion would have asthe translocation domain, a signal peptide, and then as in the case ofthe other chimeric proteins, a binding domain and an action domain. Theaction domains may be derived from the same or different proteins. Forexample, with tissue plasminogen activator, one could have the clotbinding region as one action domain and the plasminogen active site as adifferent action domain. Alternatively, one could provide enhancedblockage of homing, by having a binding protein, such as LFA-1 as oneaction domain and a selection as a second action domain. By modifyingsubunits of proteins, e.g. integrins, T-cell receptor, sIg, or the like,one could provide soluble forms of surface membrane proteins which couldbe brought together to bind to a molecule. Other opportunities arecomplement proteins, platelet membrane proteins involved in clotting,autoantigens on the surface of cells, and pathogenic molecules on thesurface of infectious agents.

[0207] IX. Introduction of Constructs into Cells

[0208] The constructs described herein can be introduced as one or moreDNA molecules or constructs, where there will usually be at least onemarker and there may be two or more markers, which will allow forselection of host cells which contain the construct(s). The constructscan be prepared in conventional ways, where the genes and regulatoryregions may be isolated, as appropriate, ligated, cloned in anappropriate cloning host, analyzed by restriction or sequencing, orother convenient means. Particularly, using PCR, individual fragmentsincluding all or portions of a functional unit may be isolated, whereone or more mutations may be introduced using “primer repair”, ligation,in vitro mutagenesis, etc. as appropriate. The construct(s) oncecompleted and demonstrated to have the appropriate sequences may then beintroduced into the host cell by any convenient means. The constructsmay be integrated and packaged into non-replicating, defective viralgenomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplexvirus (HSV) or others, including retroviral vectors, for infection ortransduction into cells. The constructs may include viral sequences fortransfection, if desired. Alternatively, the construct may be introducedby fusion, electroporation, biolistics, transfection, lipofection, orthe like. The host cells will usually be grown and expanded in culturebefore introduction of the construct(s), followed by the appropriatetreatment for introduction of the construct(s) and integration of theconstruct(s). The cells will then be expanded and screened by virtue ofa marker present in the construct. Various markers which may be usedsuccessfully include hprt, neomycin resistance, thyridine kinase,hygromycin resistance, etc.

[0209] In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, can eliminate an endogenous gene andreplace it (at the same locus or elsewhere) with the gene encoded for bythe construct using materials and methods as are known in the art forhomologous recombination. Alternatively, instead of providing a gene,one may modify the transcriptional initiation region of an endogenousgene to be responsive to the signal initiating domain. In suchembodiments, transcription of an endogenous gene such as EPO, tPA, SOD,or the like, would be controlled by administration of the ligand. Forhomologous recombination one may use either Ω or O-vectors. See, forexample, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al.,Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338,153-156.

[0210] The constructs may be introduced as a single DNA moleculeencoding all of the genes, or different DNA molecules having one or moregenes. The constructs may be introduced simultaneously or consecutively,each with the same or different markers. In an illustrative example, oneconstruct would contain a therapeutic gene under the control of aspecific responsive element (e g. NFAT), another encoding the receptorfusion protein comprising the signaling region fused to the ligandreceptor domain (e.g. as in MZF3E). A third DNA molecule encoding ahoming receptor or other product that increases the efficiency ofdelivery of the therapeutic product may also be introduced.

[0211] Vectors containing useful elements such as bacterial or yeastorigins of replication, selectable and/or amplifiable markers,promoter/enhancer elements for expression in procaryotes or eucaryotes,etc. which may be used to prepare stocks of construct DNAs and forcarrying out transfections are well known in the art, and many arecommercially available.

[0212] X. Administration of Cells and Ligands

[0213] The cells which have been modified with the DNA constructs arethen grown in culture under selective conditions and cells which areselected as having the construct may then be expanded and furtheranalyzed, using, for example, the polymerase chain reaction fordetermining the presence of the construct in the host cells. Once themodified host cells have been identified, they may then be used asplanned, e.g. grown in culture or introduced into a host organism.

[0214] Depending upon the nature of the cells, the cells may beintroduced into a host organism, e.g. a mammal, in a wide variety ofways. Hematopoietic cells may be administered by injection into thevascular system, there being usually at least about 10⁴ cells andgenerally not more than about 10¹⁰, more usually not more than about 10⁸cells. The number of cells which are employed will depend upon a numberof circumstances, the purpose for the introduction, the lifetime of thecells, the protocol to be used, for example, the number ofadministrations, the ability of the cells to multiply, the stability ofthe therapeutic agent, the physiologic need for the therapeutic agent,and the like. Alternatively, with skin cells which may be used as agraft, the number of cells would depend upon the size of the layer to beapplied to the burn or other lesion. Generally, for myoblasts orfibroblasts, the number of cells will be at least about 10⁴ and not morethan about 10⁸ and may be applied as a dispersion, generally beinginjected at or near the site of interest. The cells will usually be in aphysiologically-acceptable medium.

[0215] Instead of ex vivo modification of the cells, in many situationsone may wish to modify cells in vivo. For this purpose, varioustechniques have been developed for modification of target tissue andcells in vivo. A number of virus vectors have been developed, such asadenovirus and retroviruses, which allow for transfection and randomintegration of the virus into the host. See, for example, Dubensky etal. (1984) Proc. Natl. Acad. Sd. USA 81, 7529-7533; Kaneda et al.,(1989) Science 243, 375-378; Hiebert et al. (1989) Proc. Natl. Acad.Sci. USA 86, 3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265,17285-17293 and Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA 88,8377-8381. The vector may be administered by injection, e.g.intravascularly or intramuscularly, inhalation, or other parenteralmode.

[0216] In accordance with in vivo genetic modification, the manner ofthe modification will depend on the nature of the tissue, the efficiencyof cellular modification required, the number of opportunities to modifythe particular cells, the accessibility of the tissue to the DNAcomposition to be introduced, and the like. By employing an attenuatedor modified retrovirus carrying a target transcriptional initiationregion it desired, one can activate the virus using one of the subjecttranscription factor constructs, so that the virus may be produced andtransfect adjacent cells.

[0217] The DNA introduction need not result in integration in everycase. In some situations, transient maintenance of the DNA introducedmay be sufficient. In this way, one could have a short term effect,where cells could be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

[0218] The ligand providing for activation of the cytoplasmic domain maythen be administered as desired. Depending upon the binding affinity ofthe ligand, the response desired, the manner of administration, thehalf-life, the number of cells present, various protocols may beemployed. The ligand may be administered parenterally or orally. Thenumber of administrations will depend upon the factors described above.The ligand may be taken orally as a pill, powder, or dispersion;bucally, sublingually, injected intravascularly, intraperitoneally,subcutaneously; by inhalation or the like. The ligand (and monomericcompound) may be formulated using conventional methods and materialswell known in the art for the various routes of administration. Theprecise close and particular method of administration will depend uponthe above factors and be determined by the attending physician or humanor animal healthcare provider. For the most part, the manner ofadministration will be determined empirically.

[0219] In the event that the activation by the ligand is to be reversed,the monomeric compound may be administered or other single binding sitecompound which can compete with the ligand. Thus, in the case of anadverse reaction or the desire to terminate the therapeutic effect, themonomeric binding compound can be administered in any convenient way,particularly intravascularly, if a rapid reversal is desired.Alternatively, one may provide for the presence of an inactivationdomain (or transcriptional silencer) with a DNA binding domain. Inanother approach, cells may be eliminated through apoptosis viasignalling through Fas or TNF receptor as described elsewhere.

[0220] The particular dosage of the ligand for any application may bedetermined in accordance with the procedures used for therapeutic dosagemonitoring, where maintenance of a particular level of expression isdesired over an extended period of times, for example, greater thanabout two weeks, or where there is repetitive therapy, with individualor repeated doses of ligand over short periods of time, with extendedintervals, for example, two weeks or more. A dose of the ligand within apredetermined range would be given and monitored for response, so as toobtain a time-expression level relationship, as well as observingtherapeutic response. Depending on the levels observed during the timeperiod and the therapeutic response, one could provide a larger orsmaller dose the next time, following the response. This process wouldbe iteratively repeated until one obtained a dosage within thetherapeutic range. Where the ligand is chronically administered, oncethe maintenance dosage of the ligand is determined, one could then doassays at extended intervals to be assured that the cellular system isproviding the appropriate response and level of the expression product.

[0221] It should be appreciated that the system is subject to manyvariables, such as the cellular response to the ligand, the efficiencyof expression and, as appropriate, the level of secretion, the activityof the expression product, the particular need of the patient, which mayvary with time and circumstances, the rate of loss of the cellularactivity as a result of loss of cells or expression activity ofindividual cells, and the like. Therefore, it is expected that for eachindividual patient, even if there were universal cells which could beadministered to the population at large, each patient would be monitoredfor the proper dosage for the individual.

[0222] The subject methodology and compositions may be used for thetreatment of a wide variety of conditions and indications. For example,B- and T-cells may be used in the treatment of cancer, infectiousdiseases, metabolic deficiencies, cardiovascular disease, hereditarycoagulation deficiencies, autoimmune diseases, joint degenerativediseases, e.g. arthritis, pulmonary disease, kidney disease, endocrineabnormalities, etc. Various cells involved with structure, such asfibroblasts and myoblasts, may be used in the treatment of geneticdeficiencies, such as connective tissue deficiencies, arthritis, hepaticdisease, etc. Hepatocytes could be used in cases where large amounts ofa protein must be made to complement a deficiency or to deliver atherapeutic product to the liver or portal circulation.

[0223] The following examples are offered by way illustration and not byway limitation.

EXAMPLES

[0224] Cellular Transformations and Evaluation

Example 1

[0225] Induction of Isolated IL-2 Enhancer-Binding Transcription Factorsby Cross-Linking the CD3 Chain of the T-Cell Receptor.

[0226] The plasmid pSXNeo/IL2 (IL2-SX) (FIG. 1), which contains theplacental secreted alkaline phosphatase gene under the control of humanIL-2 promoter (−325 to +47; MCB(86) 6, 3042), and related plasmidvariants (i.e. NFAT-SX, NF B-SX, OAP/Oct1-SX, and AP-1-SX) in which thereporter gene is under the transcriptional control of the minimal IL-2promoter (−325 to −294 and −72 to +47) combined with synthetic oligomerscontaining various promoter elements (i.e. NFAT, NK B, OAP/Oct-1, andAP1, respectively), were made by three piece ligations of 1) pPL/SEAP(Berger, et al., Gene (1988) 66,1) cut with SspI and HindIII 2) pSV2/Neo(Southern and Berg J. Mol. Appl. Genet. (1982) 1, 332) cut with NdeI,blunted with Klenow, then cut with PvuI and 3) variouspromoter-containing plasmids (i.e. NFAT-C8, B-CD8, cx12lacZ-Oct1,AP1-LUCIF3H, or cx15IL2) (described below) cut with PvuI and HindIII.NFAT-CD8 contains 3 copies of the NFAT-binding site (−286 to −257; Genesand Dev. (1990) 4, 1823) and cx12lacZ-Oct contains 4 copies of theOAP/Oct-1/(ARRE-1) binding site (MCB, (1988) 8, 1715) from the humanIL-2 enhancer; B-CD8 contains 3 copies of the NF B binding site from themurine light chain (EMBO (1990) 9, 4425) and AP1-LUCIF3H contains 5copies of the AP-1 site (5′-TGA-CTCAGCGC-3′) from the metallothionenpromoter.

[0227] In each transfection, 5 μg of expression vector, pCDL-SR (MCB 8,466-72) (Tac-IL2 receptor-chain), encoding the chimeric receptorTAC/TAC/Z (TTZ) (PNAS 88, 8905-8909), was co-transfected along withvarious secreted alkaline phosphatase-based reporter plasmids (see mapof pSXNeo/IL2 in FIG. 1) in TAg Jurkat cells (a derivative of the humanT-cell leukemia line Jurkat stably transfected with the SV40 large Tantigen (Northrup, et al., J. Biol. Chem. [1993 ]). Each reporterplasmid contains a multimerized oligonucleotide of the binding site fora distinct IL-2 enhancer-binding transcription factor within the contextof the minimal IL-2 promoter or, alternatively, the intact IL-2enhancer/promoter upstream of the reporter gene. After 24 hours,aliquots of cells (approximately 10⁵) were placed in microtiter wellscontaining log dilutions of bound anti-TAC (CD25) mAb (33B3.1; AMAC,Westbrook, Me.). As a positive control and to control for transfectionefficiency, ionomycin (1 μm) and PMA (25 ng/ml) were added to aliquotsfrom each transfection. After an additional 14 hour incubation, thesupernatants were assayed for the alkaline phosphatase activity andthese activities were expressed relative to that of the positive controlsamples. The addition of 1 ng/ml FK506 dropped all activity due to NFATto background levels, demonstrating that deactivations are in the samepathway as that blocked by FK506. Each data point obtained was theaverage of two samples and the experiment was performed several timeswith similar results. See FIG. 5. The data show that with a knownextracellular receptor, one obtains an appropriate response with areporter gene and different enhancers. Similar results were obtainedwhen a MAb against the TcR complex (i.e. OKT3) was employed.

Example 2

[0228] Inhibitory Activity of the Immunosuppressant Drugs FK506 andCyclosporin A (CsA) or the Dimeric Derivative Compounds FK1012A (8),FK1012B (5), and CsA dimer (PB-1-218).

[0229] Ionomycin (1 μm) and PMA (25 ng/ml) were added to 10⁵ TAg-Jurkatcells. In addition, titrations of the various drugs were added. After 5hours the cells were lysed in mild detergent (i.e. Triton X-100) and theextracts were incubated with the β-galactosidase substrate, MUG (methylgalactosidyl umbelliferone) for 1 hour. A glycine/EDTA stop buffer wasadded and the extracts assayed for fluoresces. Each data point obtainedwas the average of two samples and the experiment was performed severaltimes with similar results. Curiously, FK1012B appears to augmentmitogen activity slightly at the highest concentration (i.e. 5 μg/ml);however, a control experiment shows that FK1012B is not stimulatory byitself. See FIG. 6.

Example 3

[0230] Activity of the Dimeric FK506 Derivative FK1012A on the ChimericFKBP12/CD3 (1FK3) Receptor.

[0231] 5 μg of the eukaryotic expression vector, pBJ5, (based on pCDL-SRwith a polylinker inserted between the 16S splice site and the poly Asite), containing the chimeric receptor (1FK3), was co-transfected with4 μg of the NFAT-inducible secreted alkaline phosphatase reporterplasmid, NFAT-SX. As a control, 5 μg of pBJ5 was used, instead of1FK3/pBJ5, in a parallel transfection. After 24 hours, aliquots of eachtransfection containing approximately 10⁵ cells were incubated with logdilutions of the drug, FK1012A, as indicated. As a positive control andto control for transfection efficiency, ionomycin (1 μm) and PMA (25ng/ml) were added to aliquots from each transfection. After anadditional 14 hour incubation, the supernatants were assayed foralkaline phosphatase activity and these activities were expressedrelative to that of the positive control samples. The addition of 2ng/ml FK506 dropped all stimulations to background levels, demonstratingthat the activations are in the same pathway as that blocked by FK506.Hence, FK506 or cyclosporin will serve as effective antidotes to the useof these compounds. Each data point obtained was the average of twosamples and the experiment was performed several times with similarresults. See FIG. 7.

Example 4A

[0232] Activity of the Dimeric FK506 Derivative, FK1012B, on theMyristoylated Chimeric CD3/FKBP12 (MZF3E) Receptor.

[0233] We have successfully demonstrated a number of approaches toligand design and syntheses, including positive results with FK506-basedHOD reagents named “FK1012”s. We have found that FK1012s achieve highaffinity, 2:1 binding stoichiometry (K_(d)(1)=0.1 nM; K_(d)(2)=0.8 nM)and do not inhibit calcineurin-mediated TCR signaling. The ligands areneither “immunosuppressive” nor toxic (up to 0.1 mM in cell culture).Similarly, we have prepared a cyclosporin A-based homodimerizing agent,“(CsA)2” which binds to the CsA receptor, cyclophilin, with 1:2stoichiometry, but which does not bind to calcineurin. Thus, likeFK1012s, (CsA)2 does not inhibit signalling pathways and is thus neitherimmunosuppressive nor toxic.

[0234] These and other of our examples of ligand-mediated proteinassociation resulted in the control of a signal transduction pathway. Inan illustrative case, this was accomplished by creating an intracellularreceptor comprised of a small fragment of Src sufficient forposttranslational myristoylation (M), the cytoplasmic tail of zeta (Z; acomponent of the B cell receptor was also used), three consecutiveFKBP12M (F3) and a flu epitope tag (E). Upon expressing the constructMZF3E (FIG. 18) in human (Jurkat) T cells, we confirmed that the encodedchimeric protein underwent FK1012-mediated oligomerization. Theattendant aggregation of the zeta chains led to signaling via theendogenous TCR-signaling pathway (FIG. 15), as evidenced by secretion ofalkaline phosphatase (SEAP) in response to an FK1012 (EC₅₀=50 nM). Thepromoter of the SEAP reporter gene was constructed to betranscriptionally activated by nuclear factor of activated T cells(NFAT), which is assembled in the nucleus following TCR-signaling.FK1012-induced signaling can be terminated by a deaggregation processinduced by a nontoxic, monomeric version of the ligand called FK506-M.

[0235] Specifically, 5 μg of the eukaryotic expression vector, pBJ5,containing a myristoylated chimeric receptor was transfected with 4 μgNFAT-SX MZE, MZF1E, MZF2E and MZF3E contain 0, 1, 2, or 3 copies ofFKBP12, respectively, downstream of a myristoylated CD3 cytoplasmicdomain (see FIG. 2). As a control, 5 μg of pBJ5 was used in a paralleltransfection. After 24 hours, aliquots of each transfection containingapproximately 10⁵ cells were incubated with log dilutions of the drug,FK1012B, as indicated. As a positive control and to control fortransfection efficiency, ionomycin (1 μm) and PMA (25 ng/ml) were addedto aliquots from each transfection. After an additional 12 hourincubation, the supernatants were assayed for alkaline phosphataseactivity and these activities were expressed relative to that of thepositive control samples. The addition of 1 ng/ml FK506 dropped allstimulations to near background levels, demonstrating that theactivations are in the same pathway as that blocked by FK506. Thisresult is further evidence of the reversibility of the subject cellactivation. Each data point obtained was the average of two samples andthe experiment was performed several times with similar results. SeeFIG. 8. The myristoylated derivatives respond to lower concentrations ofthe ligand by about an order of magnitude and activate NF-AT dependenttranscription to comparable levels, but it should be noted that theligands are different Compare FIGS. 7 and 8.

[0236] In vivo FK1012induced protein dimerization. We next wanted toconfirm that intracellular aggregation of the MZF3E receptor is indeedinduced by the FK1012. The influenza haemagglutinin epitope-tag (flu) ofthe MZF3E-construct was therefore exchanged with a different epitope-tag(flag-M2). The closely related chimeras, MZF3E_(flu) and MZF3E_(flag),were coexpressed in Jurkat T cells. Immunoprecipitation experimentsusing anti-Flag-antibodies coupled to agarose beads were performed afterthe cells were treated with FK1012A. In the presence of FK1012A (1 μM)the protein chimera MZF3E_(flag) interacts with MZEF3E_(flu) and iscoimmunoprecipitated with MZF3E_(flag). In absence of FK1012A, nocoimmunoprecipitation of MZF3E_(flu) is observed. Related experimentswith FKBP monomer constructs MZF3E_(flu) and MZF3E_(flag), which do notsignal, revealed that they are also dimerized by FK1012A. This reflectsthe requirement for aggregation observed with both the endogenous T cellreceptor and our artificial receptor MZF3E.

[0237] FK1012-induced protein-tyrosine phosphorylation. Theintracellular domains of the TCR, CD3 and zeta-chains interact withcytoplasmic protein tyrosine kinases following antigen stimulation.Specific members of the Src family (lck and/or fyn) phosphorylate one ormore tyrosine residues of activation motifs within these intracellulardomains (tyrosine activation motif, TAM). The tyrosine kinase ZAP-70 isrecruited (via its two SH2 domains) to the tyrosine phosphorylatedT-cell-receptor, activated, and is likely to be involved in the furtherdownstream activation of phospholipase C. Addition of either anti-CD3MAb or FK1012A to Jurkat cells stably transfected with MZF3E resulted inthe recruitment of kinase activity to the zeta-chain as measured by anin vitro kinase assay following immunoprecipitation of the endogenous Tcell receptor zeta chain and the MZF3E-construct, respectively. Tyrosinephosphorylation after treatment of cells with either anti-CD3 MAb orFK1012 was detected using monoclonal alpha-phosphotyrosine antibodies.Whole cell lysates were analysed at varying times after stimulation. Asimilar pattern of tyrosine-phosphorylated proteins was observed afterstimulation with either anti-CD3 MAb or FK1012. The pattern consisted ofa major band of 70 kDa, probably ZAP-70, and minor bands of 120 kDa, 62kDa, 55 kDa and 42 kDa.

[0238] Example 4(B)

[0239] Regulation of Programmed Cell Death with Immunophilin-Fas AntigenChimeras

[0240] The Fas antigen is a member of the nerve growth factor(NGF)/tumor necrosis factor (TNF) receptor superfamily of cell surfacereceptors. Crosslinking of the Fas antigen with antibodies to itsextracellular domain activates a poorly understood signaling pathwaythat results in programmed cell death or apoptosis. The Fas antigen andits associated apoptotic signaling pathway are present in most cellsincluding possibly all tumor cells. The pathway leads to a rapid andunique cell death (2 h) that is characterized by condensed cytoplasm,the absence of an inflammatory response and fragmentation of nucleosomalDNA, none of which are seen in necrotic cell death.

[0241] We have also developed a second, inducible signaling system thatleads to apoptotic cell death. Like the MZF3E pathway, this one isinitiated by activating an artificial receptor that is the product of aconstitutively expressed “responder” gene. However, the new pathwaydiffers from the first in that our HOD reagents induce the synthesis ofproducts of an endogenous pathway rather than of the product of atransfected, inducible (e.g., reporter) gene.

[0242] Gaining control over the Fas pathway presents significantopportunities for biological research and medicine. Transgenic animalscan be designed with “death” responder genes under the control ofcell-specific promoters. Target cells may then be chemically ablated inthe adult animal by administering a HOD reagent to the animal. In thisway, the role of specific brain cells in memory or cognition or immunecells in the induction and maintenance of autoimmune disorders could beassessed. Death responder genes may also be introduced into tumors usingthe human gene therapy technique developed by M. Blaese and co-workers(Culver et al, Science 256 5063 (1992): 1550-2) and then subsequentlyactivated by treating the patient with a HOD reagent (in analogy to the“gancyclovir” gene therapy clinical trials recently reported for thetreatment of brain tumors). Finally, we contemplate the coadministrationof a death-responder gene together with the therapeutic gene in thepractice of gene therapy. This would provide a “failsafe” component togene therapy. If something were to go awry (a commonly discussed concernis an integration-induced loss of a tumor suppressor gene leading tocancer), the gene therapy patient could take a “failsafe” pill thatwould kill all transfected cells. We have therefore designed a system oforthogonal oligomerizing reagents for such purposes. Thus we provide forthe use of one set of ligands and chimeric responder proteins forregulating apoptosis in the host cells, and another set for regulatingthe transcription of therapeutic genes. The ligands used for regulatingtranscription of a therapeutic or desired gene are designed (orselected) to not cross-react and initiate apoptosis.

[0243] An exemplary chimeric cDNA has been constructed consisting ofthree FKBP12 domains fused to the cytoplasmic signaling domain of theFas antigen (FIG. 19). This construct, when expressed in human Jurkatand murine D10 T cells, can be induced to dimerize by an FK1012 reagentand initiate a signaling cascade resulting in FK1012-dependentapoptosis. The LD₅₀ for FK1012A-mediated death of cells transientlytransfected with MFF3E is 15 nM as determined by a loss of reporter geneactivity (FIG. 19; for a discussion of the assay, see legend to FIG.20). These data coincide with measurements of cell death in stablytransfected cell lines. Since the stable tansfectants represent ahomogeneous population of cells, they have been used to ascertain thatdeath is due to apoptosis rather than necrosis (membrane blebbing,nucleosomal DNA fragmentation). However, the transient transfectionprotocol is more convenient and has therefore been used as an initialassay system, as described below.

Example 4(C)

[0244] Regulation of Programmed Cell Death with Cyclophilin-Fas AntigenChimeras

[0245] We have also prepared a series of cyclophilin C-Fas antigenconstructs and assayed their ability to induce (CsA)2-dependentapoptosis in transient expression assays (FIG. 20A). In addition,(CsA)2-dependent apoptosis has been demonstrated with human Jurkat Tcells stably transfected with the most active construct in the series,MC3FE (M=myristoylation domain of Src, C=cyclophilin domain,F=cytoplasmic tail of Fas, E=flu epitope tag). The cytoplasmic tail ofFas was fused either before of after 1, 2, 3, or 4 consecutivecyclophilin domains. Two control constructs were also prepared that lackthe Fas domain. In this case we observed that the signaling domainfunctions only when placed after the dimerization domains. (The zetachain constructs signal when placed either before or after thedimerization domains.) Both the expression levels of the eight signalingconstructs, as ascertained by Western blotting, and their activitiesdiffered quantitatively (FIG. 20B). The optional system has thus farproved to be MC3FE. The LF₅₀ for (CsA)2-mediated cell death with MC3FEis 18 200 nM. These data demonstrate the utility of thecyclophilin-cyclosporin interactions for regulating intracellularprotein association and illustrate an orthogonal reagent system thatwill not cross-react with the FKBP12-FK1012 system. Further, in thiscase, the data show that only dimerization and not aggregation isrequired for initiation of signal transduction by the Fas cytoplasmictail.

[0246] Mutation of the N-terminal glycine of the myristoylation signalto an alanine prevents myristoylation and hence membrane localization.We have also observed that the mutated construct (ΔMFF3E) was equallypotent as an inducer of FK1012-dependent apoptosis, indicating thatmembrane localization is not necessary for Fas-mediated cell death.

Example 5

[0247] Construction of Murine Signalling Chimeric Protein.

[0248] The various fragments were obtained by using primers described inFIG. 4. In referring to primer numbers, reference should be made to FIG.4.

[0249] An approximately 1.2 kb cDNA fragment comprising the I-E chain ofthe murine class II MHC receptor (Cell, 32, 745) was used as a source ofthe signal peptide, employing P#6048 and P#6049 to give a 70 bpSacII-XhoI fragment using PCR as described by the supplier (Promega). Asecond fragment was obtained using a plasmid comprising Tac (IL2receptor chain) joined to the transmembrane and cytoplasmic domains ofCD3 (PNAS, 88, 8905). Using P#6050/and P#6051, a 320 bp XhoI-EcoRIfragment was obtained by PCR comprising the transmembrane andcytoplasmic domains of CD3 . These two fragments were ligated andinserted into a SacII-EoRI digested pBluescript (Stratagene) to provideplasmid, SPZ/KS.

[0250] To obtain the binding domain for FK506, plasmid rhFKBP (providedby S. Schireiber, Nature (1990) 346, 674) was used with P#6052 andP#6053 to obtain a 340 bp XhoI-SalI fragment containing human FBP. Thisfragment was inserted into pBluescript digested with XhoI and SalI toprovide plasmid FK12/KS, which was the source for the FKBP12 bindingdomain. SPZ/KS was digested with XhoI, phosphatased (cell intestinalalkaline phosphatase; CIP) to prevent self-annealing, and combined witha 10-fold molar excess of the XhoI-SalI FKBP12-containing fragment fromFK12/KS. Clones were isolated that contained monomers, dimers, andtrimers of FKBP12 in the correct orientation. The clones 1FK1/KS,1FK2/KS, and 1FK3/KS are comprised of in the direction of transcription;the signal peptide from the murine MHC class II gene I-E, a monomer,dimer or trimer, respectively, of human FKBP12, and the transmembraneand cytoplasmic portions of CD3. Lastly, the SacII-EcoRI fragments wereexcised from pBluescript using restriction enzymes and ligated into thepolylinker of pBJ5 digested with SacII and EcoRI to create plasmids1FK1/pBJ5, 1FK2/pBJ5, and 1FK3/pBJ5, respectively. See FIGS. 3 and 4.

Example 6

[0251] A. Construction of Intracellular Signaling Chimera.

[0252] A myristoylation sequence from src was obtained from Pellman, etal., Nature 314, 374, and joined to a complementary sequence of CD3 toprovide a primer which was complementary to a sequence 3′ of thetransmembrane domain, namely P#8908. This primer has a SacII siteadjacent to the 5′ terminus and a XhoI sequence adjacent to the 3′terminus of the myristoylation sequence. The other primer P#8462 has aSalI recognition site 3′ of the sequence complementary to the 3′terminus of CD3, a stop codon and an EcoRI recognition site. Using PCR,a 450 bp SacII-EcoRI fragment was obtained, which was comprised of themyristoylation sequence and the CD3 sequence fused in the 5′ to 3′direction. This fragment was ligated into SacII/EcoRI-digestedpBJ5(XhoI)(SalI) and cloned, resulting in plasmid MZ/pBJ5. Lastly,MZ/pBJ5 was digested with SalI, phosphatased, and combined with a10-fold molar excess of the XhoI-SalI FKBP12-containing fragment fromFK12/KS and ligated. After cloning, the plasmids comprising the desiredconstructs having the myristoylation sequence, CD3 and FKBP12 multimersin the 5′-3′ direction were isolated and verified as having the correctstructure. See FIGS. 2 and 4.

[0253] B. Construction of Expression Cassettes for Intracellular SignalChimeras

[0254] The construct MZ/pBJ5 (MZE/pBJ5) is digested with restrictionenzymes XhoI and SalI the TCR ζ fragment is removed and the resultingvector is ligated with a 10 fold excess of a monomer, dimer, trimer orhigher order multimer of FKBP12 to make MF1E, MF2E, MF3E orMF_(n)E/pBJ5. Active domains designed to contain compatible flankingrestriction sites (i.e. XhoI and SalI) can then be cloned into theunique XhoI or SalI restriction sites of MF_(n)E/pBJ5.

Example 7

[0255] Construction of Nuclear Chimera

[0256] A. GAL4 DNA Binding Domain-FKBP Domain(s)-Epitope Tag.

[0257] The GAL4 DNA binding domain (amino acids 1-147) was amplified byPCR using a 5′ primer (#37) that contains a SacII site upstream of aKozak sequence and a translational start site, and a 3′ primer (#38)that contains a SalI site. The PCR product was isolated, digested withSacII and SalI and ligated into pBluescript II KS(+) at the SacII andSalI Sites, generating the construct pBS-GAL4. The construct wasverified by sequencing. The SacII/SalI fragment from pBS-GAL4 wasisolated and ligated into the IFK1/pBJ5 and IFK3/pBJ5 constructs(containing the myristoylation sequence, see Example 6) at the SacII andXhoI sites, generating constructs GF1E, GF2E and GE3E. 5′end of PCRamplified produce      SacII          |----GAL4 (I-147)--->>                    M  K  L  L S   S  I 5′ CGAC{overscore(ACCGCG)}GCCACCATGAAGCTACTGTCTTCTATCG              K{overscore (ozak)}3′end of PCR amplified product    <<----GAL4 (1-147----) I   R  Q  L  T  V  S 5′ GACAGTTGACTGTATCGGTCGACTGTCG3′ CTGTCAACTGACATAGCCAGCTGACAGC               {overscore ( SalI )}

[0258] B. HNF1 Dimerization/DNA Binding Domain-FKBP Domain(s)-Tag.

[0259] The HNF1a dimerization/DNA binding domain (amino acids 1-282) wasamplified by PCR using a 5′ primer (#39) that contains a SacII siteupstream of a Kozak sequence and a translational start site, and a 3′primer (#40) that contains a SalI site. The PCR product was isolated,digested with SacII and SalI, and ligated into pBluescript II KS(+) atthe SacII and SalI sites, generating the construct pBS-HNF. Theconstruct was verified by sequencing. The SacII/SalI fragment frompBS-HNF was isolated and ligated into the IFK1/pBJ5 and IFK/pBj5constructs at the SacII and XhoI sites, generating constructs HF1E, HF2Eand HF3E. 5′ end of PCR amplified productSacII           |--HNF1(1-281)-->>                 M  V  S  K  L  S5′ CGACACCGCGGCCACCATGGTTTCTAAGCTGAGC          {overscore (Kozak)} 3′endof PCR amplified product    <<-HNF1 (1-282)-1    A  F  R  H  K  L5′ CCTCCGGCACAAGTTGGTCGACTGTCC 3′ GGAAGGCCGTGTTCAACCAGCTGACAGC             {overscore ( SalI  )}

[0260] C. FKBP Domain(s)-VP16 Transcrip. Activation Domain(s)-EpitopeTag.

[0261] These constructs were made in three steps: (i) a construct wascreated from IFK3/pBJ5 in which the myristoylation sequence was replacedby a start site immediately upstream of an XhoI site, generatingconstruct SF3E; (ii) a nuclear localization sequence was inserted intothe XhoI site, generating construct NF3E; (iii) the VP16 activationdomain was cloned into the SalI site of NF3E, generating constructNF3V1E.

[0262] (i). Complementary oligonucleotides (#45 and #46) encoding aKozak sequence and start site flanked by SacII and XhoI sites wereannealed, phosphorylated and ligated into the SacII and XhoI site ofMF3E, generating construct SF3E.

[0263] Insertion of generic start site         Kozak          M  L E5′ GG{overscore (CCACC)}ATGC 3′ CGCCGGTGGTACGAGCT    {overscore(Sa)}cII        {overscore (XhoI)}    overhang     overhang

[0264] (ii). Complementary oligonucleotides (#47 and #48) encoding theSV40 T antigen nuclear localization sequence flanked by a 5′ SalI siteand a 3′ XhoI site were annealed, phosphorylated and ligated into theXhoI site of SF1E, generating the construct NF1E. The construct wasverified by DNA sequencing. A construct containing the mutant ordefective form of the nuclear localization sequence, in which athreonine is substituted for the lysine at position 128, was alsoisolated. This is designated NF1E-M. Multimers of the FKBP12 domain wereobtained by isolating the FKBP12 sequence as an XhoI/SalI fragment frompBS-FKBP12 and ligating this fragment into NF1E linearized with XhoI.This resulted in the generation of the constructs NF2E and NF3E.

[0265] Insertion of NLS into generic start site              T (ACN)          126          132       L  D  P  K  K  K  R  K  V  L  E5′ TCGACCCTAAGAAGAAGAGAAAGGTAC 3′     GGGATTCTTCTTCTCTTTCCATGAGCT  {overscore (SalI )}                        {overscore (XhoI)}

[0266] Threonine at position 128 results in a defective NLS.

[0267] (iii). The VP16 transcriptional activation domain (amino acids413-490) was amplified by PCR using a 5′ primer (#43) that contains SalIsite and a 3′ primer (#44) that contains an XhoI site. The PCR productwas isolated, digested with SalI and XhoI, and ligated into MF3E at theXhoI and SalI sites, generating the construct MV1E. The construct wasverified by sequencing. Multimerized VP16 domains were created byisolating the single VP16 sequence as a XhoI/SalI fragment from MV1E andligating this fragment into MV1E linearized with XhoI. Constructs MV2E,MV3E and MV4E were generated in this manner. DNA fragments encoding oneor more multiple VP16 domains were isolated as XhoI/SalI fragments fromMV1E or MV2E and ligated into NF1E linearized with SalI, generating theconstructs NF1V1E and NF1V3E. Multimers of the FKBP12 domain wereobtained by isolating the FKBP12 sequence as an XhoI/SalI fragment frompBS-FKBP12 and ligating this fragment into NF1V1E linearized with XhoI.This resulted in the generation of the constructs NF2V1E and NF3V1E.5′end of PCR amplified product         SalI   |--VP16(413-490)--->>              A  P  P  T  D  V  5′ CGACA{overscore(GTCGAC)}GCCCCCCCGACCGATGTC 3′end of PCR amplified product  <<--VP16(413-490)----|     D  E  Y  G  G 5′ GACGAGTACGGTGGGCTCGAGTGTCG3′ CTGCTCATGCCACCCGAGCTCACAGC               {overscore ( Xhol )}

[0268] Oligonucleotides: #37 38mer/0.2 um/OFF5′CGACACCGCGGCCACCATGAAGCTACTGTCTTCTATCG #38 28mer/0.2 um/OFF5′CGACAGTCGACCGATACAGTCAACTGTC #39 34mer/0.2 um/OFF5′CGACACCGCGGCCACCATGGTTTCTAAGCTGAC #40 28mer/0.2 um/OFF5′CGACAGTCGACCAACTTGTGCCGGAAGG #43 29mer/0.2 um/OFF5′CGACAGTCGACGCCCCCCCGACCGATGTC #44 26mer/0.2 um/OFF5′CGACACTCGAGCCCACCGTACTCGTC #45 26mer/0.2 um/OFF 5′GGCCACCATGC #4618mer/0.2 um/OFF 5′TCGAGCATGGTGGCCGC #47 27mer/0.2 um/OFF5′TCGACCCTAAGA-(C/A)-GAAGAGAAAGGTAC #48 27mer/0.2 um/OFF5′TCGAGTACCTTTCTCTTC-(G/T)-TCTTAGGG

Example 8

[0269] Demonstration of Transcriptional Induction.

[0270] Jurkat TAg cells were transfected with the indicated constructs(5 μg of each construct) by electroporation (960 μF, 250 v). After 24hours, the cells were resuspended in fresh media and aliquoted. Half ofeach transfection was incubated with the dimeric FK506 derivative,(Example 14) at a final concentration of 1 μM. After 12 hours, the cellswere washed and cellular extracts were prepared by repeated freeze-thaw.Chloramphenicol acetyltransferase (CAT) activity was measured bystandard protocols. Molecular Cloning: A Laboratory Manual, Sambrook etal. eds. (1989) CSH Laboratory, pp. 16-59 ff. The data demonstrated CATactivity present as expected (in sample 2, with or without ligand; andin samples 5 and 6 in the presence of ligand) in 70 μL of extract (totalextract volume was 120 μL) after incubation at 37° C. for 18 hours. Thesamples employed in the assays are as follows:

[0271] 1. G5E4TCAT (GAL4-CAT reporter plasmid)

[0272] 2. G5F4TCAT, GAL4-VP16

[0273] 3. G5E4TCAT, NF3V1E

[0274] 4. G5E4TCAT, GF2E

[0275] 5. G5E4TCAT, GF2E, NF3V1E

[0276] 6. G5E4TCAT, GE3E, NF3V1E

[0277] Synthetic Chemistry Examples

[0278] As indicated elsewhere, compounds of particular interest atpresent as oligomerization agents have the following structure:

linker-}rbm₁, rbm₂, . . . rbm_(n)}

[0279] wherein “linker” is a linker moiety such as described hereinwhich is covalently linked to “n” (an integer from 2 to about 5, usually2 or 3) receptor binding moieties (“rbm”'s) which may be the same ordifferent. As discussed elsewhere herein, the receptor binding moiety isa ligand (or analog thereof) for a known receptor, such as areenumerated in Section V(C), and including FK506, FK520, rapamycin andanalogs thereof which are capable of binding to an FKBP; as well ascyclosporins, tetracyclines, other antibiotics and macrolides andsteroids which are capable of binding to respective receptors.

[0280] The linker is a bi- or multi-functional molecule capable of beingcovalently linked (“-”) to two or more receptor binding moieties.Typically the linker would comprise up to about 40 atoms and may includenitrogen, oxygen and sulfur in addition to carbon and hydrogen.Illustrative linker moieties are disclosed in Section VI(A) and in thevarious Examples and include among others C1-C30 alkyl, alkylene, orarylalkyl groups which may be substituted or unsubstituted and may bestraight-chain, branched or cyclic. For example, alkyl substituents aresaturated straight-chain, cyclic or branched hydrocarbon moieties,preferably of one to about twelve carbon atoms, including methyl, ethyl,n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, t-butyl, cyclobutyl,cyclopropylmethylene, pentyl, hexyl, heptyl, octyl and so forth, and maybe optionally substituted with one or more substituents such as loweralkoxy, carboxy, amino (substituted or unsubstituted), phenyl, aryl,mercapto, halo (fluoro, chloro, bromo or iodo), azido or cyano.

[0281] These compounds may be prepared using commercially availablematerials and/or procedures known in the art. Engineered receptors forthese compounds may be obtained as described infra. Compounds ofparticular interest are those which bind to a receptor with a Kd of lessthan 10⁻⁶, preferably less than about 10⁻⁷ and even more preferably,less than 10⁻⁸ M.

[0282] One subclass of oligomerizing agents of interest are those inwhich one or more of the receptor binding moieties is FK506, anFK506-type compound or a derivative thereof, wherein the receptorbinding moieties are covalently attached to the linker moiety throughthe allyl group at C21 (using FK6 numbering) as per compound 5 or 13 inFIG. 9A, or through the cyclohexyl ring (C29-C34), e.g. through the C32hydroxyl as per compounds 8, 16, 17 in FIG. 9B. Compounds of this classmay be prepared by adaptation of methods disclosed herein, including inthe examples which follow.

[0283] Another subclass of oligomerizing agents of interest are those inwhich at least one of the receptor binding moieties is FK520 or aderivative thereof, wherein the molecules of FK520 or derivativesthereof are covalently attached to the linker moiety as in FK1040A orFK1040B in FIG. 10. Compounds of this class may be prepared byadaptation of Scheme 1 in FIG. 10, Scheme 2 in FIGS. 11A and 11B orScheme 3 in FIG. 12 and FIG. 13.

[0284] A further subclass of oligomerizing agents of interest are thosein which at least one of the receptor binding moieties is cyclosporin Aor a derivative.

[0285] It should be appreciated that these and other oligomerizingagents of this invention maybe homo-oligomerizing reagents (where therbm's are the same) or hetero-oligomerizing agents (where the rbm's aredifferent). Hetero-oligomerizing agents may be prepared by analogy tothe procedures presented herein, including Scheme 3 in FIG. 13 and asdiscussed elsewhere herein.

[0286] The following synthetic examples are intended to be illustrative.

[0287] A. General Procedures. All reactions were performed in oven-driedglassware under a positive pressure of nitrogen or argon. Air andmoisture sensitive compounds were introduced via syringe or cannulathrough a rubber septum.

[0288] B. Physical Data. Proton magnetic resonance spectra (¹H NMR) wererecorded on Bruker AM-500 (500 MHz), and AM-400 (400 MHz) spectrometers.Chemical shifts are reported in ppm from tetramethylsilane using thesolvent resonance as an internal standard (chloroform, 727 ppm). Dataare reported as follows: chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, br=broadened, m=multiplet), couplingconstants (Hz), integration. Low and high-resolution mass spectra wereobtained.

[0289] C. Chromatography. Reactions were monitored by thin layerchromatography (TLC) using E. Merck silica gel 60F glass plates (0.25mm). Components were visualized by illumination with long waveultraviolet light, exposed to iodine vapor, and/or by dipping in anaqueous ceric ammonium molybdate solution followed by heating. Solventsfor chromatography were HPLC grade. Liquid chromatography was performedusing forced flow (flash chromatography) of the indicated solvent systemon E. Merck silica gel 60 (230-400 mesh).

[0290] D. Solvents and Reagents. All reagents and solvents wereanalytical grade and were used as received with the followingexceptions. Tetrahydrofuran (THF), benzene, toluene, and diethyl etherwere distilled from sodium metal benzophenone ketyl. Triethylamine andacetonitrile were distilled from calcium hydride. Dichloromethane wasdistilled from phosphorous pentoxide. Dimethylformamide (DMF) wasdistilled from calcium hydride at reduced pressure and stored over 4Amolecular sieves.

[0291] Preparation of FK506 Derivatives

Example 9

[0292] Hydroboration/Oxidation of FK506-TBS₂ (1 to 2).

[0293] The hydroboration was performed according to the procedure ofEvans (Evans, et al., JACS (1992) 114, 6679; ibid. (1992) 6679-6685.(See Harding, et al., Nature (1989) 341, 758 for numbering.) A 10-mlflask was charged with 24,32-bis[(tert-butyldimethylsilyl)oxy]-FK506(33.8 mg, 0.033 mmol) and [Rh(nbd)(diphos-4)]BF₄(3.1 mg, 0.004 mmol, 13mol %). The orange mixture was dissolved in toluene (2.0 mL) and thesolvent was removed under reduced pressure over four hours. The flaskwas carefully purged with nitrogen and the orangish oil was dissolved inTHF (3.0 mL, 10 mM final concentration) and cooled to 0° C. with an icewater bath. Catecholborane (98 μL, 0.098 mmol, 1.0 M solution in THF,3.0 equiv.) was added via syringe and the resulting solution was stirredat 0° C. for 45 min. The reaction was quenched at 0° C. with 0.2 mL ofTHF/EtOH (1:1) followed by 0.2 mL of pH 7.0 buffer (Fisher; 0.05 Mphosphate) then 0.2 mL of 30% H₂O₂. The solution was stirred at roomtemperature for at least 12 h. The solvent was removed under reducedpressure and the remaining oil was dissolved in benzene (10 mL) andwashed with saturated aqueous sodium bicarbonate solution (10 mL). Thephases were separated and the aqueous phase was back-extracted withbenzene (2×10 mL). The organic phases were combined and washed once withsaturated aqueous sodium bicarbonate solution (10 mL). The benzene phasewas dried with MgSO_(4,) concentrated, and subjected to flashchromatography (2.1 hexane:ethyl acetate) providing the desired primaryalcohol as a clear, colorless oil (12.8 mg, 0.012 mmol, 37%).

[0294] Preparation of Mixed Carbonate (2 to 3). The preparation of themixed carbonate was accomplished by the method of Ghosh (Ghosh, et al.,Tetrahedron Lett. (1992) 33, 2781-2784). A 10-mL flask was charged withthe primary alcohol (29.2 mg, 0.0278 mmol) and benzene (4 mL). Thesolvent was removed under reduced pressure over 60 min. The oil wasdissolved in acetonitrile (2.0 mL, 14 mM final concentration) andstirred at 20° C. as triethylamine (77 μL, 0.56 mmol) was added.N,N′-disuccinimidyl carbonate (36 mg, 0.14 mmol) was added in oneportion and the solution was stirred at 20° C. for 46 h. The reactionmixture was diluted with dichloromethane and washed with saturatedaqueous sodium bicarbonate solution (10 mL). The phases were separatedand the aqueous layer was back extracted with dichloromethane (2×10 mL).The organic phases were combined and dried (MgSO₄, concentrated, andsubjected to flash chromatography (3:1 to 2:1 to 1:1 hexane:ethylacetate). The desired mixed carbonate was isolated as a clear, colorlessoil (16.8 mg, 0.014 mmol, 51%).

[0295] Dimerization of FK506 (3 to 4). A dry, 1-mL conical glass vial(Kontes Scientific Glassware) was charged with the mixed carbonate (7.3mg, 0.0061 mmol) and acetonitrile (250 μL, 25 mM final concentration).Triethylamine (10 μL, 0.075 mmol) was added followed byp-xylylenediamine (83 μL, 0.0027 mmol, 0.32 M solution in DMF). Thereaction stirred 22 h at 20° C. and was quenched by dilution withdichloromethane (10 mL). The solution was washed with saturated aqueoussodium bicarbonate solution (10 ml). The phases were separated and theaqueous layer was back-extracted with dichloromethane (2×10 mL). Theorganic phases were combined and dried (MgSO₄), concentrated, andsubjected to flash chromatography (3:1 to 2:1 to 1:1 hexane:ethylacetate) providing the desired protected dimer as a clear, colorless oil(4.3 mg, 1.9 μmol, 70%).

[0296] Deprotection of the FK506 Dimer (4 to 5). The protected dimer (33mg, 1.4 μmol) was placed in a 1.5-mL polypropylene tube fitted with aspin vane. Acetonitrile (0.5 mL, 3 mM final concentration) was added andthe solution stirred at 20° C. as HF(55 μL 48% aqueous solution; Fisher)was added. The solution was stirred 18 h at room temperature. Thedeprotected FK506 derivative was then partitioned betweendichloromethane and saturated aqueous sodium bicarbonate in a 15-mL testtube. The tube was vortexed extensively to mix the phases and, afterseparation, the organic phase was removed with a pipet. The aqueousphase was back-extracted with dichloro-methane (4×2 mL), and thecombined organic phases were dried (MgSO₄), concentrated and subjectedto flash chromatography (1:1:1 hexane:THF:ether to 1:1 THF:ether)providing the desired dimer as a clear, colorless oil (1.7 mg, 0.93μmol, 65%).

[0297] Following the above procedure, other monoamines and diamines maybe used, such as benzylamine (14) octamethylenediamine,decamethylenediamine, etc.

Example 10

[0298] Reduction of FKS506 with L-Selectride (FK506 to 6).

[0299] Danishefsky and coworkers have shown that the treatment of FK506with L-Selectride provides 22-dihydro-FK506 with a boronate esterengaging the C24 and C22 hydroxyl groups (Coleman and Danishefsky,Heterocycles (1989) 28, 157-161; Fisher, et al., J. Org. Chem. (1991)56, 2900-2907).

[0300] Preparation of the Mixed Carbonate (6 to 7). A 10-mL flask wascharged with 22-dihydro-FK506-sec-butylboronate (1253 mg, 0.144 mmol)and acetonitrile (3.0 mL, 50 mM final concentration) and stirred at roomtemperature as triethylamine (200 μL, 1.44 mmol, 10 equiv.) was added tothe clear solution. N,N′-disuccinimidyl carbonate (184.0 mg, 0.719 mmol)was added in one portion, and the clear solution was stirred at roomtemperature for 44 h. The solution was diluted with ethyl acetate (20mL) and washed with saturated aqueous sodium bicarbonate (10 mL) and thephases were separated. The aqueous phase was then back extracted withethyl acetate (2×10 mL), and the organic phases were combined, dried(MgSO₄), and the resulting oil was subjected to flash chromatography(1:1 to 1:2 hexane:ethyl acetate) providing the desired mixed carbonateas a clear, colorless oil (89.0 mg, 0.088 mmol, 61%).

[0301] Dimerization of FK506 Mixed Carbonate (7 to 8). A dry, 1-mLconical glass vial (Kontes Scientific Glassware) was charged with themixed carbonate (15.0 mg, 0.0148 mmol) and dichloromethane (500 μL, 30mM final concentration). The solution was stirred at room temperature astriethylamine (9 μL, 0.067 mmol, 10 equiv.) was added followed byp-xylylenediamine (0.8 mg, 0.0059 mmol). The reaction stirred 16 h at20° C. and was quenched by dilution with dichloromethane (5 mL). Thesolution was washed with saturated aqueous sodium bicarbonate solution(5 mL). The phases were separated and the aqueous layer was backextracted with dichloromethane (2×5 mL). The organic phases werecombined and dried (MgSO₄), concentration, and subjected to flashchromatography (1:1 to 1:2 hexane:ethyl acetate) providing the desireddimer as a clear, colorless oil (7.4 mg, 3.8 μmol, 65%).

[0302] Following the above procedure, other, monoamines, diamines ortriamines may be used in place of the xylylenediamine, such asbenzylamine (15), octylenediamine, decamethylenediamine (16),bis-p-dibenzylamine, N-methyl diethyleneamine, tris-aminoethylamine(17), tris-aminopropylamine, 1,3,5-triaminomethylcyclohexane, etc.

Example 11

[0303] Oxidative Cleavage and Reduction of FK506 (1 to 9).

[0304] The osmylation was performed according to the procedure of Kelly(VanRheenen, et al., Tetrahedron Lett. (1976) 17, 1973-1976). Thecleavage was performed according to the procedure of Danishefsky (Zell,et al., J. Org. Chem. (1986) 51, 5032-5036). The aldehyde reduction wasperformed according to the procedure of Krishnamurthy (J. Org. Chem.,(1981) 46, 4628-4691). A 10 mL flask was charged with24,32-bis[tert-butyldimethylsilyl)-FK506 (84.4 mg, 0.082 mmol),4-methylmorpholine N-oxide (48 mg, 0.41 mmol, 5 equiv), and THF (2.0 mL,41 mM final concentration). Osmium tetroxide (45 μL, 0.008 mmol 0.1equiv) was added via syringe. The clear, colorless solution was stirredat room temperature for 5 hr. The reaction was then diluted with 50%aqueous methanol (1.0 mL) and sodium periodate (175 mg, 0.82 mmol, 10equiv) was added in one portion. The cloudy mixture was stirred 40 minat room temperature, diluted with ether (10 mL), and washed withsaturated aqueous sodium bicarbonate solution (5 mL). The phases wereseparated and the aqueous layer was back-extracted with ether (2×5 mL).The combined organic layers were dried (MgSO₄) and treated with solidsodium sulfite (50 mg). The organic phase was then filtered andconcentrated and the oil was subjected to flash chromatography (3:1 to2:1 hexane:ethyl acetate) providing the intermediate, unstable aldehyde(53.6 mg) as a clear, colorless oil. The aldehyde was immediatelydissolved in THF (4.0 mL) and cooled to −78° C. under an atmosphere ofnitrogen, and treated with lithium tris[(3-ethyl-3-pentyl)oxy]aluminiumhydride (0.60 mL, 0.082 mmol, 0.14 M solution in THF, 1.0 equiv). Theclear solution was allowed to stir for 10 nun at −78° C. then quenchedby dilution with ether (4 mL) and addition of saturated aqueous ammoniumchloride (0.3 mL). The mixture was allowed to warm to room temperatureand solid sodium sulfate was added to dry the solution. The mixture wasthen filtered and concentrated and the resulting oil was subjected toflash chromatography (2:1 hexane:ethyl acetate) giving the desiredalcohol as a clear, colorless oil (39.5 mg, 0.038 mmol, 47%).

[0305] Preparation of Mixed Carbonate (9 to 10). The preparation of themixed carbonate was accomplished by the method of Ghosh, et al.,Tetrahedron Lett. (1992) 33, 2781-2784). A 10 mL flask was charged withthe primary alcohol (38.2 mg, 0.0369 mmol) and acetonitrile (2.0 mL, 10mM final concentration) and stirred at room temperature as 2,6-lutidine(43 μL, 0.37 mmol, 10 equiv) was added. N,N′-disuccinimidyl carbonate(48 mg, 0.18 mmol) was added in one portion and the solution was stirredat room temperature for 24 h. The reaction mixture was diluted withether (10 mL) and washed with saturated aqueous sodium bicarbonatesolution (10 mL). The phases were separated and the aqueous layer wasback-extracted with ether (2×10 mL). The organic phases were combinedand dried (MgSO₄), concentrated, and subjected to flash chromatography(2:1 to 1:1 hexane:ethyl acetate). The desired mixed carbonate wasisolated as a clear, colorless oil (32.6 mg, 0.028 mmol, 75%).

[0306] Preparation of Benzyl Carbamate (10 to 11). A dry, 1 mL conicalglass vial (Kontes Scientific Glassware) was charged with the mixedcarbonate 10 (8.7 mg, 0.0074 mmol) and acetonitrile (500 μL, 15 mM finalconcentration). The solution was stirred at room temperature astriethylamine (10 μL, 0.074 mmol, 10 equiv) was added followed bybenzylamine (1.6 μL, 0.015 mmol 2 equiv). The reaction stirred 4 h atroom temperature. The solvent was removed with a stream of dry nitrogenand the oil was directly subjected to flash chromatography (3:1 to 2:1hexane:ethyl acetate) providing the desired protected monomer as aclear, colorless oil (6.2 mg, 5.3 μmol, 72%).

[0307] The protected monomer (6.2 mg, 5.3 μmol) was placed in a 1.5 mLpolypropylene tube fitted with a spin vane. Acetonitrile (0.5 mL, 11 mMfinal concentration) was added and the solution stirred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.0 N finalconcentration) was added. The solution was stirred 18 h at roomtemperature. The deprotected FK506 derivative was then partitionedbetween dichloromethane and saturated aqueous sodium bicarbonate in a 15mL test tube. The tube was vortexed extensively to mix the phases and,after separation, the organic phase was removed with a pipes. Theaqueous phase was back-extracted with dichloromethane (4×2 mL), and thecombined organic phases were dried (MgSO₄), concentrated and subjectedto flash chromatography (1:1 to 0:1 hexane:ethyl acetate) providing thedesired deprotected benzylcarbamate as a clear, colorless oil (3.9 mg,4.1 μmol, 78%).

[0308] By replacing the benzylamine with a diamine such asxylylenediamine (12), hexamethylenediamine, octamethylenediamine,decamethylenediamine (13) or other diamines, dimeric compounds of thesubject invention are prepared.

Example 12

[0309] Preparation of the Mixed Carbonate of FK506 (12).

[0310] A 10-mL flask was charged with 24,32-bis[(tert-butyldimethylsilyl)oxy]-FK506 (339.5 mg., 0.329 mmol),4-methylmorpholine N-oxide (193 mg, 1.64 mmol, 5 equiv), water (0.20 mL)and THF(8.0 mL, 41 mN final concentration). Osmium tetroxide (0.183 mL,0.033 mmol 0.1 equiv, 0.18 M soln in water) was added via syringe. Theclear, colorless solution was stirred at room temperature for 4.5 h. Thereaction was diluted with 50% aqueous methonol (4.0 mL) and sodiumperiodate (700 mg, 3.29 mmol 10 equiv) was added in one portion. Thecloudy mixture was stirred 25 min at room temperature, diluted withether (20 mL), and washed with saturated aqueous sodium bicarbonatesolution (10 mL). The phases were separated and the aqueous layer wasback extracted with ether (2×10 mL). The combined organic layers weredried over MgSO₄ and solid sodium sulfite (50 mg). The organic phase wasthen filtered and concentrated and the resulting aldehyde wasimmediately dissolved in THF (8.0 mL) and cooled to −78° C. under anatmosphere of nitrogen, and treated with lithium tris[(3-ethyl-3-pentyl)oxy] aluminum hydride (2.35 mL, 0.329 mmol, 0.14 Msolution of THF, 1.0 equiv). The clear solution was allowed to stir for60 min at −78° C. (monitored closely by TLC) then quenched at −78° C. bydilution with ether (5 mL) and addition of saturated aqueous ammoniumchloride (0.3 mL). The mixture was allowed to warm to room temperatureand solid sodium sulfate was added to dry the solution. The mixture wasstirred 20 min, filtered, concentrated, and the resulting oil wasimmediately dissolved in acetonitrile (10 mL). To the solution of theresulting primary alcohol in CH₃CN was added 2,6-lutidine (0.380 mL, 3.3mmol, 10 equiv) and N,N′-disuccinimidyl carbonate (420 mg, 1.65 mmol, 5equiv). The heterogenous mixture was stirred at room temperature for 19h, at which time the solution was diluted with ether (30 mL) and washedwith saturated aqueous sodium bicarbonate (20 mL). The aqueous phase wasback-extracted with ether (2×10 mL). The organic phases were combinedand dried (MgSO₄), concentrated, and subjected to flash chromatography(3:1 to 2:1 to 1:1 hexane/ethyl acetate). The desired mixed carbonate 12was isolated as a clear, colorless oil (217 mg, 0.184 mmol, 56% overallfor 4 steps).

Example 13

[0311] Preparation of 24,24′,32,32′-tetrakis[(tert-butyldimethylsilyl)oxy]-FK1012-A. (p-xylylenediamine bridge)

[0312] A dry, 1-mL conical glass vial was charged with the mixedcarbonate (23.9 mg, 0.0203 mmol) and acetonitile (500 μl, 41 mM finalconcentration). Triethylamine (28 μL, 0.20 mmol, 10 equiv) was addedfollowed by p-xylylenediamine (46 μL, 0.0101 mmol, 0.22 M solution inDMF). The reaction stirred 18 h at room temperature, the solvent wasremoved with a stream of dry nitrogen, and the oil was directlysubjected to flash chromatography (3:1 to 2:1 to 1:1 hexane/ethylacetate) affording the desired protected dimer as a clear, colorless oil(11.9 mg, 53 μmol, 52%).

Example 14

[0313] Preparation of FK1012-A (p-xylylenediamine bridge) (13).

[0314] The protected dimer (11.0 mg, 4.9 μmol) was placed in a 1.5-mLpolypropylene tube fitted with a spin vane. Acetonitrile (0.50 mL, 10 mMfinal concentration) was added, and the solution stirred at 20° C. as HF(55 μL, 48% aqueous solution; Fisher, 3.0 N final concentration) wasadded. The solution was stirred 16h at room temperature. The deprotectedFK506 derivative was then partitioned between dichloromethane andsaturated aqueous sodium bicarbonate in a 15-mL test tube. The tube wasvortexed extensively to mix the phases and, after separation, theorganic phase was removed with a pipet. The aqueous phase wasback-extracted with dichloromethane (4×2 mL), and the combined organicphases were dried (MgSO₄), concentrated and subjected to flashchromatography (1:1:1 hexane/THF/ether to 1:1 THF/ether) providingFK1012-A as a clear, colorless oil (5.5 mg, 3.0 μmol, 63%).

Example 15

[0315] Preparation of24,24′,32,32′-tetrakis[(ter-butyldimethylsilyl)oxy]-FK1012-B(diaminodecane bridge).

[0316] A dry, 1-mL conical glass vial was charged with the mixedcarbonate (53.3 mg, 0.0453 mmol) and acetonitrile (2.0 mL, 11 m M finalconcentration). Triethylamine (16 μL, 0.11 mmol, 5 equiv) was addedfollowed by diaminodecane (61 μL, 0.0226 mmol, 0.37 M solution in DMF).The reaction stirred 12 h at room temperature, the solvent was removedwith a stream of dry nitrogen, and the oil was directly subjected toflash chromatography (3:1 to 2:1 to 1:1 hexane/ethyl acetate) affordingthe desired protected dimer as a clear, colorless oil (18.0 mg, 7.8μmol, 35%).

Example 16

[0317] Preparation of FK1012-B (diaminodecane-1,10 bridge) (14).

[0318] The protected dimer (180 mg, 7.8 μmol) was placed in a 1.5-mLpolypropylene tube fitted with a stirring flea. Acetonitrile (0.45 mL,16 mM final concentration) was added, and the solution stirred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.6 N finalconcentration) was added. The solution was stirred 17 h at 23° C. Theproduct FK1012-B was then partitioned between dichloromethane andsaturated aqueous sodium bicarbonate in a 15-mL test tube. The tube wasvortexed extensively to mix the phases and, after separation, theorganic phase was removed with a pipet. The aqueous phase wasback-extracted with dichloromethane (4×2 mL), and the combined organicphases were dried (MgSO₄, concentrated and subjected to flashchromatography (100% ethyl acetate to 20:1 ethyl acetate/methanol)affording FK1012-B as a clear, colorless oil (5.3 mg, 2.9 μmol 37%).

Example 17

[0319] Preparation of24,24′,32,32′-tetrakis[(tert-butyldimethylsilyl)oxy]-FK1012-C(bis-p-aminomethylbenzoyl diaminodecane bridge).

[0320] A dry 25-mL tear-shaped flask was charged with the diamine linker(15.1 mg, 0.0344 mmol) and 1.0 mL of DMF. In a separate flask, the mixedcarbonate and triethylamine (0.100 mL, 0.700 mmol, 20 equiv) weredissolved in 2.0 mL of dichloromethane then added slowly (4×0.5 mL) tothe stirring solution of bis-p-aminomethylbenzoyl, diaminodecane-1,10.The flask containing the mixed carbonate 12 was washed withdichloromethane (2×0.50 mL) to ensure complete transfer of the mixedcarbonate 12. The reaction stirred 16 h at 23° C., the solvent wasremoved with a stream of dry nitrogen, and the oil was directlysubjected to flash chromatography (1:1 to 1:2 hexane/ethyl acetate) toafford the desired protected dimer as a clear, colorless oil (29.6 mg,11.5 μmol, 34%).

Example 18

[0321] Preparation of FK1012-C (15).

[0322] The protected dimer (29.6 mg, 11.5 μmol) (17) was placed in a1.5-mL polypropylene tube fitted with a stirring flea. Acetonitrile(0.45 mL, 23 mM final concentration) was added, and the solution stirredat room temperature as HF (55 μL, 48% aqueous solution; Fisher, 3.6 Nfinal concentration) was added. The solution was stirred 17 h at roomtemperature. The desired symmetrical dimer was then partitioned betweendichloromethane and saturated aqueous sodium bicarbonate in a 15 mL testtube. The tube was vortexed extensively to mix the phases and, afterseparation, the organic phase was removed with a pipet. The aqueousphase was back-extracted with dichloromethane (4×2 mL), and the combinedorganic phases were dried (MgSO₄), concentrated and subjected to flashchromatography (100% ethyl acetate to 15:1 ethyl acetate/methanol)affording FK1012-C as a clear, colorless oil (11.5 mg, 5.5 μmol, 47%).

[0323] Preparation of CsA Derivatives

Example 19

[0324] MeBmt(OAc)—OH¹CsA (2). MeBmt(OAc)—OAc¹—CsA (1) (161 mg, 124 mmol)(see Eberle and Nuninger, J. Org. Chem. (1992) 57, 2689) was dissolvedin Methanol (10 mL). KOH (196 mg) was dissolved in water (8 mL). 297 μLof the KOH solution (0.130 mmol, 1.05 eq.) was added to the solution of(1) in MeOH. This new solution was stirred at room temperature under aninert atmosphere for 4 hours at which time the reaction was quenchedwith acetic acid (2 mL). The reaction mixture was purified by reversedphase HPLC using a 5 cm×25 cm, 12 μ, 100 A, C18 column at 70° C. elutingwith 70% acetonitrile/H₂O containing 0.1% (v/v) Trifluoroacetic add togive 112 mg (72%) of the desired monoacetate (2).

[0325] MeBmt(OAc)—OCIm¹CsA (3). MeBmt(OAc)—OH¹—CsA (2) (57 mg, 45.5μmol) and carbonyldiimidazole (15 mg, 2 eq., 91 μmol) were transferredinto a 50 mL round bottom flask and dissolved in dry THF (6 mL).Diisopropylethylamine (32 μL, 4 eq., 182 μmol) was added and then thesolvent was removed on a rotary evaporator at room temperatures. Theresidue was purified by flash chromatography on silica gel usingethylacetate as eluent to give 45 mg (73%) of the desired carbamate (3).

[0326] Tris-(2-aminoethyl)amine CsA Trimer Triacetate (6).MeBmt(OAc)—OCOIm¹-CsA (3) (7.5 mg, 5.54 μmol, 3.1 eq.) was dissolved inTHF (100 μL). Diisopropylethylamine (62 μL, 5 eq., 8.93 μmol of asolution containing 100 μL of amine in 4 mL THF) was added followed bytris(2-aminoethyl)amine (26 μL, 1.79 μmol, 1 eq. of a solutioncontaining 101 mg of tris-amine in 10 mL THF). This solution was allowedto stir under N₂ atmosphere for 5 days. The reaction mix was evaporatedand then purified by flash chromatography on silica gel using 0-5%methanol in chloroform to give 4.1 mg of desired product (6).

Example 20

[0327] Diaminodecane CsA Dimer (8).

[0328] Solid Na metal (200 mg excess) was reacted with dry methanol (10mL) at 0° C. Diaminodecane CsA Dimer Diacetate (5) (4.0 mg) wasdissolved in MeOH (5 mL). 2.5 mL of the NaOMe solution was added to thesolution of (5). After 2.5 hours of stirring at room temperature underan inert atmosphere, the solution was quenched with acetic acid (2 mL)and the product was purified by reversed phase HPLC using a 5 mm×25 mm,12 μ, 100 A, C18 column at 70° C. eluting with 70-95% acetonitrile/H₂Oover 20 minutes containing 0.1% (v/v) Trfifuoroacetic add to give 2.5 mg(60%) of the desired diol.

[0329] The diaminodecane CsA Dimer Diacetate (5) was prepared byreplacing the tris(2-aminoethyl)amine with 0.45 eq. of1,10-diaminodecane.

Example 21

[0330] p-Xylylenediamine CsA Dimer (4). The p-xylene diamine CsA Dimer(4) was prepared by replacing the tris(2-aminoethyl)amino with 0.45 eq.of p-xylylene diamine.

[0331] Following procedures described in the literature otherderivatives of cyclophilin are prepared by linking at a site other thanthe 1(MeBmt 1) site.

[0332] Position 8 D-isomer analogues are produced by feeding theproducing organism with the D-amino analogue to obtain incorporationspecifically at that site. See Patchett et al., J. Antibiotics (1992)45, 943 (β-MeSO)D-Ala⁸-CsA); Traber, et al., ibid. (1989) 42, 591). Theposition 3 analogues are prepared by poly-lithiation/alkylation of CsA,specifically at the carbon of Sac3. See Wenger, Transplant Proceeding(1986) 18, 213, supp. 5 (for cyclophilin binding and activity profiles,particularly D-MePhe³-CsA); Seebach, U.S. Pat. No. 4,703,033, issuedOct. 27, 1987 (for preparation of derivatives).

[0333] Instead of cyclosporin A, following the above-describedprocedures, other naturally-occurring variants of CsA may bemultimerized for use in the subject invention.

Example 21A

[0334] Alternative Synthesis for CsA Dimer

[0335] MeBmt(OH)-η-OCOIm¹-CsA

[0336] MeBmt(OH)-η-OH¹—CsA (38 mg, 31 μmol, 1218.6 g/mol) andcarbonyldiimidazole (20 mg, 4 eq., 124 μmol, 162.15 g/mol) weretransferred into a 10 mL round bottom flask and dissolved in dry THF (2mL). Diisopropylethylamine (22 μL, 4 eq., 125 μmol, 129.25 g/mol) wasadded and then the solvent was removed on a rotary evaporator at roomtemperature. The residue was purified by flash chromatography on silicagel using 0-20% acetone in ethyl acetate as eluent to give 32mg (78%yield) of a white solid.

[0337] (CsA)2 Xylylenediamine CsA Dimer

[0338] MeBmt(OH)-η-OCOIm¹-CsA (12.5 mg, 9.52 μmol, 1312.7 g/mol) wasdissolved in DCM (200 μL). To this solution was added 22 μl (0.5 eq.,4.75 μmol) of a solution of xylylene diamine (14.7 mg, 136.2g/mol) inDMSO (0.5 mL) and the reaction mixture was stirred for 72 hours at roomtemperature under a nitrogen atmosphere concentrating slowly. Thereaction was diluted with acetonitrile (2 mL) filtered through glasswool and purified by reverse phase HPLC (Beckman C18, 10 μ, 100A, 1cm×25 cm, 5 mL/min, 50 to 90% ACN/H₂O(+0.1% TFA) over 30 minutes, 70°C.) to give 6.1 mg (49% yield) of a white solid.

Example 21B

[0339] Synthesis of a FK506-CsA Dimer

[0340] MeBmt(OAc)-η-CH₂COOEt-CsA

[0341] MeBmt(OAc)-η-Br¹—CsA (26 mg, ˜80% pure, 15.7 μmol, 1323.57 g/mol)was dissolved in THF (500 μL). This solution was added by syringe pumpover 15 hours to a THF solution of the magnesium enolate of ethylhydrogen malonate (excess) prepared by the addition of iPrMgCl (2.15 mL,2.34 M in ether) to a 0° C. solution of ethyl hydrogen malonate(Lancaster, 2.5 mmol, 332 mg, 132.12 g/mol) in ThF (4.7 mL) followed bywarming to room temperature. The reaction mixture was quenced with 1NHCL (50 mL) and extracted with ethyl acetate (2×50 mL). The organiclayers were dried over Na₂SO₄ filtered and evaporated.

[0342] The crude product was dissolved in DMF (1 mL). Et₄NOAc.4H₂O (150mg, excess) was added and the mixture was heated at 90° C. for 2 hours.The reaction mixture was cooled to room temperature, diluted with H₂O(50 mL) and extracted with ether (2×50 mL). The combined organics weredried over Na2SO4, filtered and evaporated. The residue was purified byflash chromatography on silica gel, eluting with 75-100% ethylacetate/hexanes to give 11.4 mg (55%) of a white solid.

[0343] MeBmt(OH)-η-CH₂COOH¹—CsA

[0344] MeBmt(OAc)-η-CH₂COOEt¹-CsA (11.0 mg, 8.27 μmol, 1330.76 gl/mol)was dissolved in MeOH (2 mL) and added to a solution of NaOMe (1.30 M inMeOH, 10 mL). The reaction mixture was stirred at room temperature undera nitrogen atmosphere for 5 hours at which time H₂O (2 mL) was added andthe mixture was stirred for another 2 hours. The reaction was quencedwith glacial acetic acid (1 mL), filtered through glass wool andpurified by reverse phase HPLC (Rainin C18 dynamax, 5 μ, 300A, 21.4mm×250 mm, 20 mL/min, 50 to 90% ACN/H₂O (+0.1% TFA) over 30 minutes, 70°C.) to give 5.5 mg (53% yiel) of a white solid.

[0345] bis-TBS-N-(6-(Boc-amino)hexyl) FKS506 Carbamate

[0346] bis-TBS-FKS506 succinimidyl carbonate (also a precursor to(tbs)4-FK1012) (5.8 mg, 1177.62 g/mol, 4.93 μmol) was dissolved in DCM.To this was added N-Boc-1.6-diaminohexane (7.25 mg, excess). Afterstirring for 10 min at room temperature the reaction mixture wasevaporated and the product purified by flash chromatography eluting with10 to 40% ethyl acetate/hexanes to provide 5.9 mg (94% yield).

[0347] N-(6-aminohexyl) FKS506 Carbamate

[0348] bis-TBS-N-(6(Boc-amino)hexyl) FK506 carbamate (59 mg, 1278.88g/mol, 4.61 μmol) was transfered to a polypropylene tube in ACN (700 μl)followed by aqueous HF (49%, 100 μL). The reaction was complete aftersix hours at room temperature and was quenched by the slow addition of asaturated solution of NaHCO₃. The mixture was diluted with saturatedNaHCO₃ (4 mL), H2O (4 mL) and extracted with DCM (3×10 mL). The combinedorganic phases were dried with MgSO₄, filtered and evaporated to give3.6 mg (82% yield) of crude product.

[0349] FKCsA

[0350] MeBmt(OH)-η-CH₂COOH¹—CsA (2.86 mg, 2.27 μmol, 1260.66 g/mol) andN-(6-aminohexyl) FK506 carbamate (crude, 2.16 mg, 2.28 μmol, 949.21g/mol) were dissolved in DCM (900 μL). To this solution was added 127 μl(3.0 eq., 6.8 μmol) of a solution of BOP (11.9 mg, 442.5 g/mol) in DCM(500 μL), followed by 45 μL (2.25 eq., 5.1 μmol) of a solution ofdiisopropylethyl amine (20 μL, d=0.74 2129.25 g/mol) in DCM (1.0 mL).Finally DMF (40 μL) was added and the reaction mixture was evaporatedslowly at room temperature under a stream of nitrogen over 12 hours. Thereaction mixture was diluted with acetonitrile (1 mL) filtered throughglass wool and purified by reverse phase HPLC (Becktnan C18, 1 cm×25cm,5 mL/min, 50 to 90% ACN/H₂O over 25 minutes, 50° C.) to give 2.4 mg (48%yield) of a white solid.

Example 22

[0351] (A) Structure-Based Design and Synthesis of FK1012-“Bump”Compounds and FKBP12s with Compensatory Mutations

[0352] Substituents at C9 and C10 of FK506, which can be and have beenaccessed by synthesis, clash with a distinct set of FKBP12 sidechainresidues. Thus, one class of mutant receptors for such ligands shouldcontain distinct modifications, one creating a compensatory hole for theC10 substituent and one for the C9 substituent. Carbon 10 wasselectively modified to have either an N-acetyl or N-formyl groupprojecting from the carbon (vs. a hydroxyl group in FK506). The bindingproperties of these derivatives clearly reveal that these C10 bumpseffectively abrogate binding to the native FKBP12. FIG. 21 depictssyntheses of FK506-type moieties containing additional C9 bumps. Byassembling such ligands with linker moieties of this invention one canconstruct HED and HOD (and antagonist) reagents for chimeric proteinscontaining corresponding binding domains bearing compensatory mutations.An illustrative HED reagent is depicted in FIG. 21 that containsmodifications at C9 and C10.

[0353] This invention thus encompasses a class of FK506-type compoundscomprising an FK506-type moiety which contains, at one or both of C9 andC10, a functional group comprising —OR, —R, —(CO)OR, —NH(CO)H or—NH(CO)R, where R is substituted or unsubstituted, alkyl or arylalkylwhich may be straight-chain, branched or cyclic, including substitutedor unsubstituted peroxides, and carbonates. “FK506-type moieties”include FK506, FK520 and synthetic or naturally occurring variants,analogs and derivatives hereof (including rapamycin) which retain atleast the (substituted or unsubstituted) C2 through C15 portion of thering structure of FK506 and are capable of binding with a natural ormodified FKBP, preferably with a Kd value below about 10⁻⁶ M.

[0354] This invention further encompasses homo- and hetero-dimers andhigher order oligomers containing one or more of such FK506-typecompounds covalently linked to a linker moiety of this invention.Monomers of these FK506-type compounds are also of interest, whether ornot covalently attached to a linker moiety or otherwise modified withoutabolishing their binding affinity for the corresponding FKBP. Suchmonomeric compounds may be used as oligomerization antagonist reagents,i.e., as antagonists for oligomerizing reagents based on a likeFK506-type compound. Preferably the compounds and oligomers comprisingthem in accordance with this invention bind to natural, or preferablymutant, FKBPs with an affinity at least 0.1% and preferably at leastabout 1% and even more preferably at least about 10% as great as theaffinity of FK506 for FKBP12. See e.g. Holt et al, infra.

[0355] Receptor domains for these and other ligands of this inventionmay be obtained by structure-based, site-directed or random mutagenesismethods. We contemplate a family of FKBP12 moieties which contain Val,Ala, Gly, Met or other small amino acids in place of one or more ofTyr26, Phe36, Asp37, Tyr82 and Phe99 as receptor domains for FK506-typeand FK520-type ligands containing modifications at C9 and/or C10. Inparticular, we contemplate using FKBP's with small replacements such asGly or Ala for Asp37 in conjunction with FK506-type and FK520-typeligands containing substituents at C10 (e.g., —NHCOR, where R is alkyl,preferably lower alkyl such as methyl for example; or —NHCHO), andFKBP's with small replacements such as Gly or Ala for Phe36, Phe99 andTyr26 in conjunction with FK506-type and FK520-type ligands containingreplacements at C9 (e.g., oxazalines or imines).

[0356] Site-directed mutagenesis may be conducted using the megaprimermutagenesis protocol (see e.g., Sakar and Sommer, BioTechniques 8 4(1990): 404-407). cDNA sequencing is performed with the Sequenase kit.Expression of mutant FKBP12s may be carried out in the plasmid pHN1⁺ inthe E. coli strain XA90 since many FKBP12 mutants have been expressed inthis system efficiently. Mutant proteins may be conveniently purified byfractionation over DE52 anion exchange resin followed by size exclusionon Sepharse as described elsewhere. See e.g. Aldape et al, J Biol Chem267 23 (1992): 16029-32 and Park et al, J Biol Chem 267 5 (1992):3316-3324. Binding constants may be readily determined by one of twomethods. If the mutant FKBPs maintain sufficient rotamase activity, thestandard rotamase assay may be utilized. See e.g., Galat et al.Biochemistry 31 (1992): 2427-2434. Otherwise, the mutant FKBP12s may besubjected to a binding assay using LH20 resin and radiolabeled³H₂-dihydroFK506 and ³H₂-dihyroCsA that we have used previously withFKBPs and cyclophilins. Bierer et al, Proc. Nat. Aca. Sci. U.S.A. 87 4(1993): 555-69.

[0357] (B) Selection of Compensatory Mutations in FKBP12 for Bump-FK506sUsing the Yeast Two-Hybrid System

[0358] One approach to obtaining variants of receptor proteins ordomains, including of FKBP12, is the yeast “two-hybrid” or “interactiontrap” system. The two-hybrid system has been used to detect proteinsthat interact with each other. A “bait” fusion protein consisting of atarget protein fused to a transcriptional activation domain isco-expressed with a cDNA library of potential “hooks” fused to aDNA-binding domain. A protein-protein (bait-hook) interaction isdetected by the appearance of a reporter gene product whose synthesisrequires the joining of the DNA-binding and activation domains. Theyeast two-hybrid system mentioned here was originally developed byElledge and coworkers. Durfee et al, Genes & Development 7 4 (1993):555-69 and Harper et al, Cell 75 4 (1993): 805-816.

[0359] Since the two-hybrid system per se cannot provide insights intoreceptor-ligand interactions involving small molecule organic ligands,we have developed a new, FK1012-inducible transcriptional activationsystem (discussed below). Using that system one may extend the twohybrid system so that small molecules (e.g., FK506s or FK1012s orFK506-type molecules of this invention) can be investigated. One firstgenerates a cDNA library of mutant FKPs (the hooks) with mutations thatare regionally localized to sites that surround C9 and C10 of FK506. Forthe bait, two different strategies may be pursued the first uses theability of FK506 to bind to FKBP12 and create a composite surface thatbinds to calcineurin. The sequence-specific transcriptional activator isthus comprised of: DNA-binding domain-mutantFKBP12-bump-FK506-calcineurin A-activation domain (where—refers to anoncovalent binding interaction). The second strategy uses the abilityof FK1012s to bind two FKBPs simultaneously. A HED version of an FK1012may be used to screen for the following ensemble: DNA-bindingdomain-mutant FKBP12-bump-FK506-normal FK506-wildtype FKBP12-activationdomain.

[0360] 1. Calcineurin-GAL4 activation domain fusion as a bait: Aderivative of pSE1107 that contains the GAL4 activation domain andcalcineurin. A subunit fusion construct has been constructed. Itsability to act as a bait in the proposed manner has been verified bystudies using the two-hybrid system to map out calcineurin's FKBP-FK506binding site.

[0361] 2. hFKBP12-GAL4 activation domain fusion as a bait: hFKBP12 cDNAmay be excised as an EcoRI-HindIII fragment that covers the entire openreading frame, blunt-ended and ligated to the blunt-ended XhoI site ofpSE1107 to generate the full-length hFKBP-GAL4 activation domain proteinfusion.

[0362] 3. Mutant hFKBP12 cDNA libraries hFKBP12 may be digested withEcoRI and HindIII, blunted and cloned into pAS1 (Durfee et al, supra)that has been cut with NcoI and blunted. This plasmid is furtherdigested with NdeI to eliminate the NdeI fragment between the NdeI sitein the polylinker sequence of pAS1 and the 5′ end of hFKBP12 andreligated. This generated the hFKBP12-GAL4 DNA binding domain proteinfusion. hFKBP was reamplified with primers #11206 and #11210, PrimerTable: 11206             NdeI  5NdFK: 5′-GGAATTTC {overscore (CATATG)} GGC GTG CAG G-3′          H   M   G   V   Q 11207        SmaI  3SmFK37: 5′-CTG{overscore (TC CCG G)}GA NNN NNN NNN TTT CTT TCC ATC TTCAAG C-:        R   S   X   X   X   K   K   G   D   E   L 11208       SmaI   3SmFK27: 5′-CTG{overscore (TC CCG G)}GA GGA ATC AAA TTTCTT TCC ATC TTC AAG CA⁻       R   S   S   D   F   K   K   G   D   E   L   M NNN NNN NNN GTG CACCAC GCA GG-3′  X   X   X   H   V   V   C 11209        BamHI  38mFK98:5′-CGC {overscore (GGA TCC)} TCA TTC CAG TTT TAG AAG CTC CAC ATC NNN            END  E   L   K   L   L   E   V   D   X NNN NNN AGT GGC ATGTGG-3′  X   X   T   A   H   P 11210        BamHI  38mFK: 5′-CGC{overscore (GGA TCC)} TCA TTC CAG TTT TAG AAG C-3′             END  E   L   K   L   L Primer Table: Primers used in theconstruction of a regionally localized hFKBP12 cDNA library for use inscreening for compensatoy mutations.

[0363] Mutant hFKBP12 cDNA fragments were then prepared using theprimers listed below that contain randomized mutant sequences of hFKBPat defined positions by the polymerase chain reaction, and were insertedinto the GAL4 DNA binding domain-hFKBP(NdeI/BamHI) construct.

[0364] 4. Yeast strain S. cerevisiae Y153 carries two selectable markergenes (his3/β-galactosidase) that are integrated into the genome and aredriven by GAL4 promoters. (Durfee, supra.).

[0365] Using Calcineurin GAL4 Activation Domain as Bait. TheFKBP12-FK506 complex binds with high affinity to calcineurin, a type 2Bprotein phosphatase. Since we use C9- or C10-bumped ligands to serve asa bridge in the two-hybrid system, only those FKBPs from the cDNAlibrary that contain a compensatory mutation generate a transcriptionalactivator. For convenience, one may prepare at least three distinctlibraries (using primers 11207-11209, Primer Table) that will eachcontain 8,000 mutant FKBP12s. Randomized sites were chosen by inspectingthe FKBP12-FK506 structure, which suggested clusters of residues whosemutation might allow binding of the offending C9 or C10 substituents onbumped FK506s. The libraries are then individually screened using bothC9- and C10-bumped. The interaction between a bumped-FK506 and acompensatory hFKBP12 mutant can be detected by the ability of host yeastto grow on his dropout medium and by the expression of β-galactosidasegene. Since this selection is dependent on the presence of thebumped-FK506, false positives can be eliminated by substractivescreening with replica plates that are supplemented with or without thebumped-FK506 ligands.

[0366] Using hFKBP12GAL4 Activation Domain as Bait Using the calcineurinA-GAL4 activation domain to screen hFKBP12 mutant cDNA libraries is asimple way to identify compensatory mutations on FKBP12. However,mutations that allow bumped-FK506s to bind hFKBP12 may disrupt theinteraction between the mutant FKBP12-bumped-FK506 complex andcalcineurin. If the initial screening with calcineurin as a bait fails,the wild type hFKBP12-GAL4 activation domain will instead be used. AnFK1012 HED reagent consisting of native-FK506-bumped-FK506 (FIG. 16) maybe synthesized and used as a hook. The FK506 moiety of the FK1012 canbind the FKBP12-GAL4 activation domain. An interaction between thebumped-FK506 moiety of the FK1012 and a compensatory mutant of FKBP12will allow host yeast to grow on this drop-out medium and to expressβ-galactosidase. In this way, the selection is based solely on theability of hFKBP12 mutant to interact with the bumped-FK506. The samesubstractive screening strategy can be used to eliminate falsepositives.

[0367] In addition to the in vitro binding assays discussed earlier, anin vivo assay may be used to determine the binding affinity of thebumped-FK506s to the compensatory hFKBP12 mutants. In the yeasttwo-hybrid system, β-gal activity is determined by the degree ofinteraction between the “bait” and the “prey”. Thus, the affinitybetween the bumped-FK506and the compensatory FKBP12 mutants can beestimated by the corresponding β-galactosidase activities produced byhost yeasts at different HED (native-FK506-bumped-FK506) concentrations.

[0368] Using the strategy, additional randomized mutant FKBP12 cDNAlibraries may be created in other bump-contact residues withlow-affinity compensatory FKBP12 mutants as templates and may bescreened similarly.

[0369] Phage Display Screening for High-Affinity Compensatory FKBPMutations

[0370] Some high-affinity hFKBP12 mutants for bump-FK506 may containseveral combined point mutations at discrete regions of the protein. Thesize of the library that contains appropriate combined mutations can betoo large for the yeast two-hybrid systems capacity (e.g., >10⁸mutations). The use of bacteriophage as a vehicle for exposing wholefunctional proteins should greatly enhance the capability for screeninga large numbers of mutations. See e.g. Bass et al, Proteins: Structure,Function & Genetics 8 4 (1990): 309-14; McCafferty et al, Nature 3486301 (1990): 552-4; and Hoogenboom, Nucl Acids Res 19 15 (1991): 4133-7.If the desired high-affinity compensatory mutants is not be identifiedwith the yeast two-hybrid system, a large number of combined mutationscan be created on hFKBP12 with a phage vector as a carrier. The mutanthFKBP12 fusion phages can be screened with bumped-FK506-Sepharose as anaffinity matrix, which can be synthesized in analogy to our originalFK506-based affinity matrices. Fretz et al, J Am Chem Soc 113 4 (1991):1409-1411. Repeated rounds of binding and phage amplification shouldlead to the identification of high-affinity compensatory mutants.

[0371] (C) Synthesis of “Bumped (CsA)2s”: Modification of MeVal(11)CsA

[0372] As detailed above, we have demonstrated the feasibility of usingcyclophilin as a dimerization domain and (CsA)2 as a HOD reagent in thecontext of the cell death signaling pathway. However, to furtheroptimize the cellular activity of the (CsA)2 reagent one may rely uponsimilar strategies as described with FK1012s. Thus, modified (bumped)CsA-based oligomerizing reagents should be preferred in applicationswhere it is particularly desirable for the reagent to be able todifferentiate its target, the artificial protein constructs, fromendogenous cyclophilins.

[0373] One class of modified CsA derivatives of this invention are CsAanalogs in which (a) NMeVal11 is replaced with NMePhe (which maybesubstituted or unsubstituted) or NMeThr (which may be unsubstituted orsubstituted on the threonine betahydroxyl group) or (b) the pro-S methylgroup of NMeVal11 is replaced with a bulky group of at least 2 carbonatoms, preferably three or more, which may be straight branched and/orcontain a cyclic moiety, and may be alkyl (ethyl, or preferably propyl,butyl, including t-butyl, and so forth), aryl, or arylalkyl. Thesecompounds include those CsA analogs which contain NMeLeu, NMeIIe, NMePheor specifically the unnatural NMe[betaMePhe], in place of MeVal11. The“(b)”CsA compounds are of formula 2 where R represents a functionalgroup as discussed above.

[0374] This invention further encompasses homo- and hetero-dimers andhigher order oligomers containing one or more such CsA analogs.Preferably the compounds and oligomers comprising them in accordancewith this invention bind to natural, or preferably mutant, cyclophilinproteins with an affinity at least 0.1% and preferably at least about 1%and even more preferably at least about 10% as great as the affinity ofCsA for cyclophilin.

[0375] A two step strategy may be used to prepare the modified[MeVal¹¹]CsA derivatives starting from CsA. In the first step theresidue MeVal11 is removed from the macrocycle. In the second step aselected amino acid is introduced at the (former) MeVal11 site and thelinear peptide is cyclized. The advantage of this strategy is the readyaccess to several modified [MeVal¹¹]CsA derivatives in comparison with atotal synthesis. The synthetic scheme is as follows:

[0376] To differentiate the amide bonds, an N,O shift has been achievedbetween the amino and the hydroxyl groups from MeBmt1 to give IsoCsA(Ruegger et al, Helv Chim Acta 59 4 (1976): 1075-92) (see scheme above).The reaction was carried out in THF in the presence of methanesulfonicadd. (Oliyai et al, Pharm Res 9 5 (1992): 617-22). The free amine wasprotected with an acetyl group with pyridine and acetic anhydride in aone-pot procedure. The overall yield of the N-acetyl protected IsoCsA is90%. The ester MeBmt1-MeVal11 bond is then reduced selectively in thepresence of the N-methyl amide bonds, e.g. using DIBAL-H. The resultingdiol is then transformed to the corresponding di-ester with anotheracid-induced N,O shift. This will prepare both the N-acetyl group andMeVal11 residues for removal through hydrolysis of the newly formedesters with aqueous base.

[0377] After protection of the free amino group the new amino acidresidue is introduced e.g. with the PyBrop coupling agent. Deprotectionand cyclization of the linear peptide with BOP in presence of DMAP(Alberg and Schireiber, Science 262 5131 (1993): 248-250) completes thesynthesis of 2. The binding of bumped-CsAs to cyclophilins can beevaluated by the same methods described for FK506s and FK1012s. Oncecyclophilins are identified with compensatory mutations, bumped (CsA)2HED and HOD reagents may be synthesized according to the methodsdiscussed previously. Of particular interest are bumped CsA compoundswhich can for dimers which themselves can bind to a cyclophilin proteinwith 1:2 stoichiometry. Homo dimers and higher order homo-oligomers,heterodimers and hetero-higher order oligomers containing at least onesuch CsA or modified CsA moiety may be designed and evaluated by themethods developed for FK1012A and (CsA)2, and optimize the linkerelement in analogy to the FK1012 studies.

[0378] Mutant cyclophilins that bind our position 11 CsA variants (2) byaccommodating the extra bulk on the ligand may be new be prepared.Cyclophilins with these compensatory mutations may be identified throughthe structure-based site-directed and random mutagenesis/screeningprotocols described in the FK1012 studies.

[0379] It is evident from the above results, that the subject method andcompositions provide for great versatility in the production of cellsfor a wide variety of purposes. By employing the subject constructs, onecan use cells for therapeutic purposes, where the cells may remaininactive until needed, and then be activated by administration of a safedrug. Because cells can have a wide variety of lifetimes in a host,there is the opportunity to treat both chronic and acute indications soas to provide short- or long-term protection. In addition, one canprovide for cells which will be directed to a particular site, such asan anatomic site or a functional site, where therapeutic effect may beprovided.

[0380] Cells can be provided which will result in secretion of a widevariety of proteins, which may serve to correct a deficit or inhibit anundesired result, such as activation of cytolytic cells, to inactivate adestructive agent, to kill a restricted cell population, or the like. Byhaving the cells present in the host over a defined period of time, thecells may be readily activated by taking the drug at a dose which canresult in a rapid response of the cells in the host. Cells can beprovided where the expressed chimeric receptor is intracellular,avoiding any immune response due to a foreign protein on the cellsurface. Furthermore, the intracellular chimeric receptor proteinprovides for efficient signal transduction upon ligand binding,apparently more efficiently than the receptor binding at anextracellular receptor domain.

[0381] By using relatively simple molecules which bind to chimericmembrane bound receptors, resulting in the expression of products ofinterest or inhibiting the expression of products, one can provide forcellular therapeutic treatment. The compounds which may be administeredare safe, can be administered in a variety of ways and can ensure a veryspecific response so as not to upset homeostasis.

[0382] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0383] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

We claim:
 1. A DNA construct encoding a chimeric protein comprising (a)at least one receptor domain capable of binding to a selected ligand,and (b) a heterologous protein domain capable of initiating apoptosis ina cell containing said chimeric protein following exposure of the cellto the ligand, said ligand being capable of binding to two or morechimeric protein molecules.
 2. A DNA construct of claim 1 wherein thechimeric protein further comprises an intracellular targeting domaincapable of directing the chimeric protein to a desired cellularcompartment.
 3. A DNA construct of claim 2 wherein the intracellulartargeting domain comprises a secretory leader sequence, a membranespanning domain, a membrane binding domain or a sequence directing theprotein to associate with vesicles or with the nucleus.
 4. A DNAconstruct of claim 1 wherein the chimeric protein has a K_(d) value forbinding to the selected ligand of less than or equal to about 10⁻⁶ M. 5.A DNA construct of claim 1 wherein the selected ligand is less thanabout 5 kDa in molecular weight.
 6. A DNA construct of claim 1 whereinthe heterologous protein domain comprises the cytoplasmic domain ofhuman Fas or a human TNFα receptor.
 7. A DNA construct of claim 1wherein the chimeric protein is capable of binding to an FK506-typeligand, a cyclosporin A-type ligand, tetracycline or a steroid ligand.8. A DNA vector containing a DNA construct of claim 1 and a selectablemarker permitting transfection of the DNA construct into host cells andselection of transfectants containing the construct.
 9. A DNA vector ofclaim 8 wherein the vector is a viral vector.
 10. A viral vector ofclaim 9 which is an adeno-, adeno associated- or retroviral vector. 11.A chimeric protein encoded by a DNA construct of claim
 1. 12. A cellcontaining and capable of expressing at least one DNA construct ofclaim
 1. 13. A cell of claim 12 characterized by the ability to becomeapoptotic and die following contact with the selected ligand.
 14. A cellof claim 12 which is a mammalian cell.
 15. A cell of claim 13 whichfurther contains (a) a DNA construct encoding a chimeric proteincomprising (i) at least one receptor domain capable of binding to asecond selected ligand and (ii) another protein domain, heterologouswith respect to the receptor domain, but capable, upon oligomerizationwith one or more other like domains, of triggering the activation oftranscription of a target gene under the transcriptional control of atranscriptional control element responsive to said oligomerization; and(b) a target gene under the expression control of a transcriptionalcontrol element responsive to said oligomerization; and which is capableof expressing the target gene following exposure of the cell to saidsecond selected ligand.
 16. A cell of claim 13 which contains a seriesof DNA constructs encoding (a) a first additional chimeric proteincontaining a DNA-binding domain and at least one receptor domain capableof binding to a first selected ligand moiety; and (b) a secondadditional chimeric protein containing a transcriptional activatingdomain and at least one receptor domain capable of binding to a secondselected ligand (which may be the same or different from the firstselected ligand moiety); and and a second DNA construct encoding atarget gene under the transcriptional control of a heterologoustranscriptional control sequence which binds with the DNA-binding domainand is responsive to the transcriptional activating domain; which cellexpresses the target gene following exposure to a substance containingthe selected ligand moiety(ies).
 17. The use, to prepare apharmaceutical composition for ablating a population of geneticallyengineered cells of claim 13, of a ligand capable of initiatingapoptosis witn said cells, said ligand having the formula: linker-{rbm₁,rbm₂, . . . rbm_(n)} wherein n is an integer from 2 to about 5,rbm₍₁₎₋rbm_((n)) are receptor binding moieties which may be the same ordifferent and which are capable of binding to the chimeric protein(s),said rbm moieties being covalently attached to a linker moiety which isa bi- or multi-functional molecule capable of being covalently linked(“-”) to two or more rbm moieties.
 18. The use, of claim 17, of a ligandto prepare a pharmaceutical composition for ablating a population ofgenetically engineered cells wherein the ligand has a molecular weightless than about 5 kDa.
 19. The use, of claim 17, of a ligand to preparea pharmaceutical composition for ablating a population of geneticallyengineered cells wherein the ligand comprise an FK506-type moiety, acyclosporin-type moiety, a steroid or tetracycline.
 20. The use, ofclaim 17, of a ligand to prepare a pharmaceutical composition forablating a population of genetically engineered cells wherein the ligandbinds to a naturally occurring receptor with a Kd value greater thanabout 10⁻⁵ M.
 21. The use, of claim 17, of a ligand to prepare apharmaceutical composition for ablating a population of geneticallyengineered cells wherein the ligand comprises a molecule of FK506,FK520, rapamycin or a derivative thereof modified at C9, C10 or both.22. The use, of claim 20, of a ligand to prepare a pharmaceuticalcomposition for ablating a population of genetically engineered cellswherein the ligand contains a linker moiety comprising a C2-C20alkylene, C4-C18 azalkylene, C6-C24 N-alkylene azalkylene, C6-C18arylene, C8-C24 ardialkylene or C8-C36 bis-carboxamido alkylene moiety.23. A method for ablating a population of genetically engineered cellsof claim 13 which comprises exposing the cells to a ligand, capable ofbinding to the chimeric protein, in an amount effective to result ininitiating cell death.
 24. A method of claim 23 wherein the cells aregrown in a culture medium and the exposing is effected by adding theligand to the culture medium.
 25. A method of claim 23 wherein the cellsare present within a host organism and the exposing is effected byadministering the ligand to the host organism.
 26. A method of claim 25wherein the host organism is a mammal and the ligand is administered byoral, bucal, sublingual, transdermal, subcutaneous, intramuscular,intravenous, intra-joint or inhalation administration in an appropriatevehicle therfor.
 27. A method for producing a cell which may beselectively killed which comprises introducing a DNA construct of claim1 into a host cell.
 28. A method of claim 27 which further comprisesselecting those cells containing the introduced DNA construct.
 29. A kitwhich comprises at least one DNA construct of claim
 1. 30. A kit ofclaim 29 which further comprises a ligand to which one or more of thechimeric proteins encoded by the DNA construct(s) bind.
 31. A kit ofclaim 29 which further comprises a monomeric ligand reagent as anantagonist for ligand-chimeric protein binding.
 32. A host organismcontaining a cell of any of claim
 12. 33. A host organism of claim 32which is a plant or animal organism.
 34. An animal of claim 33 which isa worm, insect or mammal.
 35. A mammal of claim 34 which is a mouse orother rodent or a human.